Therapeutic and diagnostic methods dependent on CYP2A enzymes

ABSTRACT

A method of regulating the activity of human cytochrome P450 isozyme CYP2A6 to control nicotine metabolism or decrease to production of carcinogens from procarcinogens, such as those present in tobacco smoke, in an individual by selectively inhibiting CYP2A6. Various prophylactic (i.e., prevention and treatment) compositions and methods are also described, including an improved oral nicotine composition and method comprising the use of nicotine together with an inhibitor of the CYP2A6 enzyme. 
     Furthermore, it has been discovered that the presence in an individual of a mutant allele of human cytochrome P450 enzyme CYP2A6 (referred to throughout this specification as “CYP2A6” for brevity) is predictive of an individual who: (i) has a decreased risk of becoming a smoker, (ii) will smoke less if he/she becomes dependent, and/or (iii) may be at relatively lower risk for cancer due to both decreased smoke exposure and decreased CYP2A6-mediated activation of tobacco smoke and other procarcinogenic substrates. This invention provides diagnostic methods for predicting tobacco dependence risk and risk for cancers related to CYP2A6 substrates in an individual by analysing for the presence of a mutant genotype for human cytochrome P450 enzyme CYP2A6 in an individual, ranging from gene duplication (multiple copies of CYP2A6) to single or even no copies due to null alleles or gene deletion.

This application is a continuation application of U.S. patentapplication Ser. No. 10/815,995 filed on Apr. 2, 2004 now abandoned,which is a divisional application of U.S. patent application Ser. No.09/584,669 filed on Jun. 1, 2000 now U.S. Pat. No. 6,908,631 which is anational stage of international application No. PCT/CA98/10193 filed onDec. 1, 1998, which claims the benefit of provisional patentapplications U.S. 60/067,020 filed on Dec. 1, 1997, U.S. 60/067,021filed on Dec. 1, 1997, U.S. 60/084,847 filed on May 8, 1998 and U.S.60/107,392 filed on Nov. 6, 1998. The contents of all of theabove-mentioned applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The invention relates to methods and compositions for regulatingnicotine metabolism in an individual; methods and compositions forenhancing nicotine replacement therapies; methods and compositions fordiagnosing tobacco risk dependence and risk for cancers and methods fortreating or preventing cancer.

REVIEW OF THE ART

Nicotine is one of the most widely used psychoactive drugs in the world.The World Health Organization reports that there are currently in excessof 1 billion smokers worldwide, or roughly one-third of the globalpopulation aged 15 years and older. It is well established that smokingis associated with a higher incidence of many diseases, includingvarious types of cancers, respiratory diseases, cardiovascular diseases,gastrointestinal disorders, as well as many other medical complications(Lee et al., Arch. Intern. Med., “Cigarette smoking, nicotine addiction,and its pharmacologic treatment,” 153(1): 34-48 (1993)).

Nicotine is the primary compound present in tobacco that is responsiblefor establishing and maintaining tobacco dependence (Henningfield etal., J. Pharmacol. Exp. Ther., “Abuse liability and pharmacodynamiccharacteristics of intravenous and inhaled nicotine,” 234(1): 1-12(1985)). Specifically, it has been established in the art that dependentsmokers adjust their smoking behaviour to maintain central nicotinelevels (McMorrow M J, et al., “Nicotine's role in smoking: an analysisof nicotine regulation,” Psychological Bulletin, 93(2):302-27 (1983);Russell M S H, “Nicotine intake and its regulation by smokers. Tobaccosmoking and nicotine,” Advances in behavioural biology, Martin W R, etal., New York, Plenum Press, 31:25-50 (1987)). It has been furtherestablished that: (i) smoking increases if nicotine content incigarettes is decreased (Benowitz N L, “Drug Therapy. PharmacologicAspects of Cigarette Smoking and Nicotine Addiction,” New Engl. J. Med.,319(20): 1318-30 (1988)), (ii) smoking increases if nicotine excretionis increased by urine acidification (Benowitz N L, “The Use of BiologicFluid Samples in Assessing Tobacco Smoke Consumption,” NIDA Res.Monogr., 48:6-26 (1983)), and (iii) smoking decreases withadministration of nicotine via concurrent I.V. or patch nicotine(Benowitz, N L et al., “Nicotine Metabolic Profile in Man: Comparison ofCigarette Smoking and Transdermal Nicotine”, J. Pharmacol. Exp. Ther.,268(1):296-303 (1994); and Benowitz, N L et al., “Intravenous NicotineReplacement Suppresses Nicotine Intake From Cigarette Smoking”, J.Pharmacol. Exp. Ther., 254(3):1000-5 (1990)).

In light of the key role of nicotine in producing tobacco dependence andregulating smoking behaviour, it is important to understand the patternof nicotine metabolism and the sources of variation of this metabolismin humans.

In humans, 60-85% of nicotine is metabolized to the inactive metabolitecotinine (Benowitz, et al. 1994). The cytochrome P450 (CYP) system hasbeen implicated in the metabolism of nicotine. Evidence for CYPinvolvement in nicotine metabolism has come from rat liver studies inwhich reconstituted purified CYPs, and specific antibodies were shown toinhibit nicotine metabolism. In particular, rat studies have shown thatphenobarbital inducible CYPs (i.e., the CYPs; -2B1, -2B2, -2C6, and-3A2) are involved in nicotine metabolism. Of 12 human CYPs formstested, CYP2B6 showed the highest nicotine oxidase activity while CYP2E1and CYP2C9 showed intermediate levels (Flammang et al., “Nicotinemetabolism by cDNA-expressed human cytochrome P-450s,” Biochem. Arch.,8:1-8 (1992)). cDNA studies have implicated CYP2B6, CYP2C9, CYP2D6 andCYP2E1 and have provided a possible role for CYP2A6 in nicotinemetabolism in isolated expression systems (Flammang et al., 1992;McCracken et al., “Cotinine formation by cDNA-expressed humancytochromes P450,” Med. Sci. Res., 20:877-878 (1992)).

In copending International patent application Ser. No. PCT/CA97/00506(filed Jul. 17, 1997), the contents of which are hereby incorporated byreference, the present inventors teach that the genetically polymorphicCYP2A6 enzyme is the major enzyme responsible for this metabolicconversion. In human populations there is considerable interindividualvariability in hepatic CYP2A6 function measured in vivo and in vitro(Yamano S, et al., “The CYP2A3 gene product catalyzes coumarin7-hydroxylation in human liver microsomes,” Biochemistry, 29:1322-1329(1990); Cholerton S, et al., “Comparison of a novel thin-layerchromatographic-fluorescence detection method with a spectrofluorometricmethod for the determination of 7-hydroxycoumarin in human urine,”Journal of Chromatography, 575(2):325-30 (1992); Rautio A, et al.,“Interindividual variability of coumarin 7-hydroxylation in healthyvolunteers,” Pharmacogenetics 2(5):227-33 (1992); and Iscan et al.,“Interindividual variability of coumarin 7-hydroxylation in a Turkishpopulation,” Eur. J. Clin. Pharmacol. 47(4):315-318 (1994)).

Tobacco products are vehicles for the delivery of nicotine to thebloodstream which quickly carries nicotine to the brain and otherorgans. Nicotine produces many physiological and behavioural effects,including alteration of brain chemistry and function, which leads to anindividual's dependence on nicotine. Dependent smokers adjust theirsmoking behaviour to regulate nicotine in the brain and body. Evidenceincludes increased smoking if nicotine content in cigarettes isdecreased (Benowitz 1988), increased smoking if nicotine excretion isincreased by urine acidification (Benowitz 1983), and decreased smokingwith concurrent I.V. or patch nicotine (Benowitz, et al. 1994; BenowitzN L, et al. 1990).

While the art has made strides in gaining an understanding of thepattern of nicotine metabolism and the sources of variation of thismetabolism in humans, there is still room for improvement. One areawhich has received little or no attention is in the diagnosis of risksfor smoking and tobacco-related cancers, for example in non-smokers ofrelatively young age. In particular, it would be desirable to have ameans by which it would be possible to readily identify individuals who:(i) have a decreased risk of becoming smokers, (ii) smoke less if theybecome dependent, and/or (iii) may be at relatively lower risk forcancer due to both decreased smoke exposure and decreasedenzyme-mediated activation of tobacco smoke procarcinogens.

Other than nicotine dependence as a result of tobacco use, nicotineitself is not considered hazardous, namely it is not considered to be acausative agent in cancer and heart and lung disease. It is the otherproducts which are found in tobacco products which are considered to beharmful, including combustion products such as carbon monoxide, gasesand tar.

Nicotine replacement therapies (also referred to throughout thisdisclosure as “NRT's”) are used to deliver nicotine to individuals in anattempt to assist an individual in abstaining from tobacco products.Recently, in a United Nations Conference on Trade and Development,entitled “Roundtable on Social and Economic Aspects of Reduction ofTobacco Smoking by Use of Alternative Nicotine Delivery Systems”, Sep.22-24, 1997, in an attempt to reduce tobacco-related morbidity andmortality, it was recommended that nicotine replacement therapies bemade more easily available than tobacco.

Smoking tobacco products amount to a rapid delivery mechanism ofnicotine to the bloodstream since almost all of the nicotine absorbedfrom tobacco smoke reaches systemic circulation without the need toinitially pass through liver. For this reason, conventional nicotinereplacement therapies have been based on the use of a delivery system(e.g., transdermal, etc.) which will systemically deliver nicotine.

Unfortunately, current commercially available NRT's are relativelyinconvenient to use and administer, and are not liked by many patients.For example, transdermal (e.g., transdermal, chewing gum, etc.) NRT'sare associated with occasional skin irritation and chewing gum (andother buccal delivery systems) NRT's are perceived as having a badtaste. Further, transdermal and chewing gum NRT's are plagued by thedelivery of inconsistent nicotine levels to the patient. Still further,alternative delivery NRT systems such as inhalers and nasal sprays havefailed to achieve patient acceptability.

Of note is that, to the knowledge of the inventors, an oral nicotinereplacement therapy is not currently commercially available. While notwishing to be bound by any particular theory or mode of action, thereason for this is believed to be as follows. Oral nicotine must firstpass through the liver before entering the systemic circulation. As aresult, extensive metabolism of nicotine occurs. In particular, oralnicotine is about 60-85% metabolized from nicotine to continue by theliver so only 15-40% of oral nicotine reaches the systemic circulation(Benowitz, et al., “Stable isotope studies of nicotine kinetics andbioavailability,” Clin. Pharmacol. Ther., 49(3):270-7 (1991); Svensson,“Clinical pharmacokinetics of nicotine,” Clin. Pharmacokinet.,12(I):30-40 (1987); and Zins, et al., “Pharmacokinetics of nicotinetartrate after single-dose liquid enema, oral, and intravenousadministration,” J. Clin. Pharmacol., 37(5):426-36 (1997)). Because thefirst-pass metabolism of nicotine is so effective and highconcentrations of nicotine can not be used without irritating thedigestive system, oral administration (e.g., a pill) has, heretofore,been an ineffective delivery system for nicotine. In light of this,there is no known effective oral nicotine replacement therapy. It wouldbe desirable to have such a therapy since it would be much moreconvenient for the patient and would be more precisely controlled by thephysician (e.g., prescribing dosage based on body weight and relatedfactors which are difficult to take into account when prescribingnicotine patch or chewing gum).

SUMMARY OF THE INVENTION

The present inventors have found that variation in nicotine metabolismamong individuals is due to variable expression of CYP2A isozymes;CYP2A6 has been shown to be the major nicotine metabolizing enzyme inhuman livers. Coumarin, a specific CYP2A6 substrate, was found tospecifically and selectively inhibit nicotine metabolism to cotinine by84%±11% in test livers, and addition of orphenadrine (a CYP2B6inhibitor) enhanced the inhibition. Methoxsalen and tranylcypromine havealso been found to be potent inhibitors of CYP2A6 and thus of nicotineto cotinine metabolism. The data indicate that variability in CYP2A6expression results in inter-individual variation in nicotine metabolism,which in turn, can have behavioural consequences such as smoking more orless cigarettes. Therefore, inhibitors of CYP2A6 can be used to regulatenicotine metabolism, and in particular substantially decrease nicotinemetabolism, thereby affecting tobacco use.

Broadly stated, the present invention relates to the diagnosis,prophylaxis and treatment of conditions requiring a reduction in theactivity of a human cytochrome P450 enzyme CYP2A (referred to as “CYP2A”for brevity). The term “CYP2A” as used herein means all isoforms ofCYP2A including but not limited to CYP2A(CYP1), CYP2A6, CYP2A7, CYP2A12,CYP2A13 and CYP2A16. Preferably the enzyme is CYP2A6.

The inventors have determined that the presence in an individual of amutant allele of human cytochrome P450 enzyme CYP2A6 (referred tothroughout this specification as “CYP2A6” for brevity) is predictive ofan individual who: (i) has a decreased risk of becoming a smoker, (ii)will smoke less if he/she becomes dependent, and/or (iii) may be atrelatively lower risk for cancer due to both decreased smoke exposureand decreased CYP2A6-mediated activation of tobacco smoke and otherprocarcinogenic substrates.

In one embodiment, this invention provides a diagnostic method fortobacco dependence risk and for cancers related to CYP2A6 substrates inan individual by analysing a DNA-containing bodily sample from theindividual for the presence of a mutant allele of human cytochrome P450enzyme CYP2A6. Preferably this method comprises genotype assaying thebodily sample, which may be genomic DNA isolated from peripheralleukocytes in the bodily sample. Alternatively the method comprisesphenotype assaying the bodily sample, which may be a fluid, such as ablood sample or blood plasma. This invention also provides diagnostickits for use in the analysis. The invention also provides a diagnosticmethod for tobacco dependence risk and for cancers related to humancytochrome P450 enzyme CYP2A6 substrates in an individual byadministering a dose of a CYP2A6 substrate to the individual anddetermining in a bodily sample from the individual the level of saidCYP2A6 substrate or a metabolite of said CYP2A6 substrate.

The invention specifically demonstrates that individuals who are carryCYP2A6 deficient alleles are less likely to become smokers and willsmoke less cigarettes if tobacco-dependent. In addition, because CYP2A6is known to activate procarcinogens, such as those found intobacco-smoke, the diagnostic aspect of the invention will be useful foridentifying the contribution of this polymorphic locus to the geneticrisk of an individual for cancer.

If the result of the diagnostic assay is that the individual possesseswild-type CYP2A6 (i.e., the individual contains no mutant alleles ofCYP2A6), the present diagnostic method and kit is predictive of anindividual who: (i) has an increased risk of becoming a smoker, (ii)will smoke more if he/she becomes dependent, and/or (iii) may be atrelatively higher risk for cancer due to both decreased smoke exposureand decreased enzyme mediated activation of procarcinogens. Once thisindividual is identified, he/she may be treated prophylactively witheffective quantities of CYP2A6 inhibitors described in detail incopending International patent application Ser. No. PCT/CA97/00506(filed Jul. 17, 1997) and U.S. provisional patent application Ser. No.60/067,021 (filed on Dec. 1, 1997), which lead to other aspects of thepresent invention.

Thus, the invention also provides a smoking prevention composition or asmoking regulation composition comprising a CYP2A6 inhibitor, togetherwith a carrier therefor, along with methods for preventing or regulatingsmoking by administering a CYP2A6 inhibitor to an individual. Likewise,this invention provides methods for cancer prevention or treatment orthe regulation of the formation of carcinogens by administering a CYP2A6inhibitor to an individual. Compositions containing a CYP2A6 inhibitorare also provided for use in these methods.

This invention provides methods for enhancing oral nicotine therapy,such as oral administration of nicotine bitartrate, by inhibitingnicotine metabolism through selective inhibition of CYP2A6, optionallywith further selective inhibition of CYP2B6. Preferred inhibitors ofCYP2A6 include coumarin, methoxsalen and tranylcypromine.

This method may be used to treat a condition requiring nicotineadministration, preferably by administering a CYP2A6 inhibitor takentogether with an oral formulation of nicotine, optionally alsoadministering a CYP2B6 inhibitor. Preferred inhibitors of CYP2A6 includecoumarin, methoxsalen and tranylcypromine.

The present inventors have surprisingly found that several naturalproducts, are inhibitors of the enzyme CYP2A. Accordingly, the presentinvention provides a method of inhibiting CYP2A comprising administeringan effective amount of a natural product or an extract of a naturalproduct to an individual in need thereof, this method being useful intreating conditions requiring regulation of CYP2A activity. In oneembodiment, the natural product is Hypericum or a Hypericum extract. Inanother embodiment, the natural product is Cichorium intybus orBougainvllra spectabillis or an extract thereof.

This invention also provides a composition comprising an oralformulation of nicotine and a CYP2A6 inhibitor, optionally alsocontaining a CYP2B6 inhibitor. Preferred inhibitors of CYP2A6 includecoumarin, methoxsalen and tranylcypromine.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. Aspects of thisinvention may be more fully deacribed in one or more of U.S. ProvisionalPatent Applications Nos. 60/067,20; 60/067,021; 60/084,847; and60/107,392, which are each incorporated herein by reference in theirentirety. It should be understood, however, that the detaileddescription and the specific examples while indicating preferredembodiments of the invention are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1 this figure shows the application of a method to characterizeCYP2A6 activity in human subjects by taking plasma samples at times asearly as 35 minutes after administration of an oral dose of coumarin(100 mg in this example). Lower doses of coumarin may also be used andthe data can also be expressed as a ratio of coumarin/7-OH coumarin. Thetest can also be performed using oral nicotine or another CYP2A6substrate.

FIG. 2A-2D show chemical structures of some representative CYP2A6inhibitors;

FIG. 3 is a graph illustrating a correlation between fasted morning andnon-fasted afternoon coumarin (C) testing sessions;

FIG. 4 is a graph showing a time course of total 7-hydroxycoumarinconcentration detected in the plasma of subjects given 100 mg ofcoumarin;

FIGS. 5 and 6 illustrate results of a study described in Example 1;

FIG. 7 illustrates mean plasma nicotine concentrations in the studyreported in Example 8;

FIG. 8 illustrates current desire to smoke in the study reported inExample 8;

FIG. 9 illustrates mean breath carbon monoxide in the study reported inExample 9;

FIG. 10 illustrates the ratio of increased plasma nicotine to increasedbreath carbon monoxide in Example 9;

FIG. 11 illustrates mean number of cigarettes consumed during thesmoking period in Example 9;

FIG. 12 illustrates the mean number of cigarette puffs taken in each10-min. period during the smoking period in Example 9;

FIG. 13 illustrates the mean latency period between the first twocigarettes in Example 9;

FIG. 14 illustrates the mean grams of tobacco burned in Example 9.

FIG. 15 illustrates the effect of CYP2A6 inhibitors methoxsalen andtranylcypromine on increasing the bioavailability of nicotine suppliedorally, with concommitant reduction in the desire to smoke.

FIG. 16 is a graph illustrating the effect of Hypericum extracts onnicotine metabolism by expressed human cDNA CYP2A6.

FIG. 17 is a graph showing the mean plasma concentration of nicotineversus time, in the presence of St. John's Wort or a placebo.

FIG. 18 is a bar graph showing the mean plasma concentration of nicotinein the presence of St. John's Wort or a placebo.

FIG. 19 are graphs illustrating effect of esculetin on nicotinemetabolism by human liver micorsomes.

FIG. 20 shows the chemical structure of various compounds found innatural products.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Broadly stated, the present invention relates to the diagnosis,prophylaxis and treatment of conditions requiring a reduction in theactivity of a CYP2A enzyme. The term “CYP2A” as used herein means allisoforms of CYP2A including but not limited to CYP2A(CYP1), CYP2A6,CYP2A7, CYP2A12, CYP2A13 and CYP2A16. Preferably the enzyme is CYP2A6.

As described in copending International patent application Ser. No.PCT/CA97/00506, the contents of which are hereby incorporated byreference, inhibition of CYP2A6 (and optionally CYP2B6) inhibits themetabolism of nicotine. In particular, it was found that CYP2A6 is amajor nicotine metabolizing enzyme in human livers and that byinhibiting CYP2A6 the metabolism of nicotine to continine in the liveris inhibited.

Diagnostic Methods

The present inventors have shown that individuals who carry CYP2A6mutant alleles (i) have a decreased risk of becoming a smoker, (ii) willsmoke less if he/she becomes dependent and/or (iii) may be at relativelylower risk for cancer due to both decreased smoke exposure and decreasedCYP2A6-mediated activation of tobacco smoke and other procarcinogenicsubstrates.

Accordingly, the present invention provides a method for determining therisk of an individual becoming a smoker comprising determining thegenotype or phenotype of a CYP2A allele in the individual wherein thepresence of a mutant allele is predictive of a decreased risk ofsmoking. Preferably, the CYP2A enzyme is CYP2A6.

Tobacco smoke contains a number of tobacco-specific procarcinogennitrosamines, for example the N-nitrosodiethylamine and4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These compoundsare termed pro- or pre-carcinogens, as they are activated by the body.Specifically, these tobacco smoke procarcinogens can be activated byCYP2A6 (Crespi, et al., “Human cytochrome P450IIA3: cDNA sequence, roleof the enzyme in the metabolic activation of promutagens, comparison tonitrosamine activation by human cytochrome P450IIE1,” Carcinogenesis11(8):1293-1300 (1990); Yamazaki, et al., “Cytochrome P450 2E1 and 2A6enzymes as major catalysts for metabolic activation ofN-nitrosodialkylamines and tobacco-related nitrosamines in human livermicrosomes,” Carcinogenesis 13(10):1789-94 (1992)). Thereforeindividuals who have CYP2A6 null alleles may also be less efficient atbioactivating tobacco smoke procarcinogens to carcinogens. This is ofparticular interest as ethnic variation in frequencies for CYP2A6variant alleles exist (Nowak et al., 1998; Fernandez-Salguero P, et al.,“A genetic polymorphism in coumarin 7-hydroxylation: sequence of thehuman CYP2A genes and identification of variant CYP2A6 alleles,” Am. J.Hum. Genet., 57(3):651-60 (1995); Yoloi and Kamataki, 1998) and may berelated to the ethnic differences in lung cancer incidence and histology(Groeger et al., 1997). Thus, individuals carrying CYP2A6 defectivealleles may have a decreased risk of developing tobacco-related cancersand other medical complications for three reasons. 1) They have adecreased risk of becoming a smoker. 2) If they do becometobacco-dependent, they smoke less than those without impaired nicotinemetabolism resulting in lower exposures to tobacco-relatedprocarcinogens (Law, et al., “The dose-response relationship betweencigarette consumption, biochemical markers and risk of lung cancer,” Br.J. Cancer 75(11):1690-1693 (1997)). 3) They may activate fewertobacco-related procarcincogens. These three factors suggest asignificant reduction in tobacco-related cancers for carriers of aCYP2A6 defective allele(s).

Accordingly, the present invention provides a method for determining therisk of an individual for developing cancer comprising determining thegenotype or phenotype of a CYP2A allele in the individual wherein thepresence of a mutant allele is predictive of a decreased risk ofdeveloping cancer. Preferably, the CYP2A enzyme is CYP2A6.

The diagnostic aspect of this invention includes both phenotypic andgenotypic methods for determining whether an individual has wild-type ormutant alleles for CYP2A6. The phenotypic assay may be performed by ametabolic study which is in effect an in vivo enzyme assay for CYP2A6activity. This assay may be performed by administering a dose of aCYP2A6 substrate, for example nicotine or coumarin, and monitoring thephysiological levels of the substrate and/or the product of enzymaticmetabolism of the substrate in the individual at one or more time pointsduring and subsequent to administration of the test dose. Typically, thelevels will be measured in a biological fluid, such as blood, plasma, orurine, using well known assays for the particular components, examplesof which are disclosed herein. An example of an in vivo phenotype andenzyme activity assay is provided in Example 3 below. This phenotypicassay can be used to classify individuals based on their normallyexpressed level of CYP2A6, which will correspond generally with thegenotype of the individual as homozygous for fully active CYP2A6,heterozygous or homozygous for a lower activity allele, in decreasingorder of nicotine metabolic rate.

The diagnostic aspect is also based on analysing a DNA-containing bodilysample from the individual for the presence of a mutant allele of humancytochrome P450 enzyme CYP2A6. As used throughout this specification,the term “mutant allele” is meant to encompass any allele havingdecreased or absent CYP2A6 activity, i.e., including null alleles. Thepresence of the mutant allele of CYP2A6 can be determined byconventional genotyping or phenotyping assays.

Many CYP2A6 alleles have been identified including, but not limited to,the wild-type allele (referred to throughout this specification as“CYP2A6*1”), and two defective or null mutant alleles (“CYP2A6*2” and“CYP2A6*3”, respectively (see, Fernandez-Salguero, et al. 1995), thecontents of which are hereby incorporated by reference). The CYP2A6*2allele differs from the wild-type allele by a single point mutationwhich leads to a leucine to histidine amino acid change at codon 1609.In vitro and in vivo studies have demonstrated that this allele is anull allele. Mutations in the CYP2A6*3 allele occur in exons 3, 6, and8. Very recently an additional CYP2A6 allele was identified whichconsists of an entire CYP2A6 gene deletion (Nunoya K et al., 1998 “A newdeleted allele in the human cytochrome P450 2A6 (CYP2A6) gene found inindividuals showing poor metabolic capacity to coumarin and(+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrocholoride(SM-12502). Pharmacogenetics 1998, 8:239-249. Of course, additionalmutant alleles which encode CYP2A6 enzymes with reduced activity may befound in individuals identified by the phenotypic and/or genotypicmethods of this invention, and these individuals will also be expectedto have lower risk of developing cancer and decreased risk of smoking.

The individual contemplated for the diagnostic methods of this invention(as well as the prophylactic and therapeutic methods described below)may be any type of mammal, but is preferably a primate, and morepreferably a human.

Preferably, the bodily sample is a fluid such as blood or blood plasma.Alternatively, the bodily sample can be tissue. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory Press (1989), the contents of which arehereby incorporated by reference, for discussion of general assaytechniques useful with the diagnostic methods described herein.

With reference to FIG. 1, there is illustrated the result of CYP2A6activity in a group (Group I) of individuals having heterozygous CYP2A6activity (i.e., each individual in this group had a single mutant alleleof CYP2A6 and a single active allele of CYP2A6) and a group ofindividuals (Group II) having wild type CYP2A6 activity (i.e. eachindividual in this group had two active alleles of CYP2A6). Blood plasmasamples from each of the individuals in both groups were takepost-administration of coumarin (100 mg) at 35 minutes, 45 minutes and75 minutes. Coumarin 7-hydroxylation was used to assess the complimentactivity of CYP2A6. The results of the phenotyping assay clearly showthat the Group I individuals have a significantly lower CYP2A6 activity(less than half at 35 and 45 minutes) than the Group II individuals.

Alternatively, the subject is an individual having a CYP2A6 genotypeassociated with an active form of the enzyme. The CYP2A6 genotype of anindividual and the existence of an active CYP2A6 enzyme in an individualmay be determined using procedures using techniques described herein.For example, coumarin 7-hydroxylation has been used to measure CYP2A6activity (see, Cholerton, et al. (1992) and Rautio, et al. (1992)).

The recognition by the present inventors that CYP2A6 is the majornicotine metabolizing enzyme in human livers suggests that the enzymecan be assayed in an individual to determine the individual's risk ofdeveloping tobacco dependence. Determination of CYP2A6 levels may alsobe used to select and monitor in an individual appropriate conventionalnicotine replacement therapies such as the nicotine patch and nicotinegum. It is unlikely that conventional nicotine replacement therapies(e.g. nicotine gum, nicotine patch, spray, pulmonary inhalation or otherforms) will have a high success outcome if an individual has high levelsof CYP2A6, although such individuals may be good candidates for enhancedNRT according to the methods described herein. Conversely, if anindividual has very low levels of CYP2A6, administering nicotine at highdosages will likely result in increased toxicity, and side effects.Furthermore, the co-administration of a CYP2A6 inhibitor with anexisting NRT would be expected to decrease the kinetics of nicotine fromthat source and to enhance the efficacy of the NRT (discussed belowunder Therapeutic Methods).

Prophylactic and Therapeutic Methods

As mentioned previously, the present invention relates to methods forthe prophylaxis and treatment of conditions requiring a reduction in theactivity of a CYP2A enzyme. In particular, the prophylactic/therapeuticaspect of the present invention relates to treatment and prevention ofsmoking, in vivo carcinogen formation and cancer in an individual. Eachof this involves administration to an individual of a CYP2A inhibitor,preferably a CYP2A6 inhibitor.

In one aspect, the present invention provides a method of preventing,treating or regulating smoking in an individual comprising administeringan effective amount of one or more substances selected from the groupconsisting of (i) substances which inhibit CYP2A activity; (ii)substances which inhibit transcription, translation of the gene encodingCYP2A, or both; (iii) substances which delete all or a portion of thegene encoding CYP2A. Preferably, the CYP2A is CYP2A6.

As used throughout this specification, the terms “smoking prevention”and “preventing smoking”, as used throughout this specification, areintended to mean that the likelihood of the onset of smoking (i.e., theprogression from a cigarette to regular smoking) in a currentnon-smoking individual (i.e., a person who has never smoked or is aex-smoker) and the return to smoking of a previous smoker (i.e. relapseprevention) is substantially mitigated.

The terms “smoking regulation” and “regulating smoking”, as usedthroughout this specification, are intended to mean that the amountsmoked by a current smoking individual is reduced or, at least, fails toincrease.

The terms “smoking treatment” or “treatment of smoking” means thestopping of all smoking or the reduction in amount of smoking asreflected in less use of tobacco products, a decrease in pattern of useor a decrease in tobacco smoke exposure. The measure of tobacco smokeexposure can be measured by analyzing breath carbon monoxide.

In another aspect, the present invention provides a method of regulatingthe formulation of a carcinogen in an individual comprisingadministering an effective amount of one or more substances selectedfrom the group consisting of (i) substances which inhibit CYP2Aactivity; (ii) substances which inhibit transcription, translation ofthe gene encoding CYP2A, or both; (iii) substances which delete all or aportion of the gene encoding CYP2A. Preferably, the CYP2A is CYP2A6.

The terms “carcinogen formation regulation” and “regulating formation ofa carcinogen”, as used throughout this specification, are intended tomean that the occurrence of carcinogen formation in an individual isreduced. This may be achieved, for example, by using CYP2A6 inhibitionto inhibit activation of procarcinogens present in the individual. Asused throughout this specification, the term “procarcinogen” is meant toencompass any substance which is at least one of procytotoxic,promutagenic and progenotoxic (“pro” means the metabolite is more activethat the parent compound).

In a further aspect, the present invention provides a method ofpreventing cancer in an individual comprising administering an effectiveamount of one or more substances selected from the group consisting of(i) substances which inhibit CYP2A activity; (ii) substances whichinhibit transcription, translation of the gene encoding CYP2A, or both;(iii) substances which delete all or a portion of the gene encodingCYP2A. Preferably, the CYP2A is CYP2A6.

The terms “cancer prevention” and “preventing cancer”, as usedthroughout this specification, are intended to mean that the likelihoodof the onset of cancer in a current cancer-free individual (i.e., aperson who has never had cancer or whose cancer is in remission) issubstantially mitigated.

The terms “inhibitor” and “inhibition”, in the context of the presentinvention, are intended to have a broad meaning and encompass substanceswhich directly or indirectly (e.g., via reactive intermediates,metabolites and the like) act on CYP2A to inhibit or otherwise regulatethe ability of CYP2A to catalyze metabolism of a substrate. Othersubstances which act indirectly on CYP2A include those substances whichinhibit transcription and/or translation of the gene encoding CYP2A. Inparticular, the terms “CYP2A6 inhibition” and “CYP2A6 inhibitor” areintended to have a broad meaning and encompass any substance which: (i)inhibits CYP2A6 activity; (ii) inhibits transcription and/or translationof the gene encoding CYP2A6; or (iii) deletes or removes the geneencoding CYP2A6. Particularly preferred substances are those which alterthe kinetics for metabolism of nicotine to cotinine, alter smokingbehavior, alter the likelihood of addiction to smoking in a populationof non-smokers, or alter the kinetics of formation for carcinogens whoseformation from procarcinogens is catalyzed by CYP2A, and more preferablyexhibit the biological altering effect without producing otherbiological effects at significant levels.

A substance will “selectively” inhibit CYP2A activity when the substancecan alter the kinetics for metabolism of nicotine to cotinine, altersmoking behavior, alter the likelihood of addiction to smoking in apopulation of non-smokers, or alter the kinetics of formation forcarcinogens whose formation from procarcinogens is catalyzed by CYP2Agenerally at a dosage level which is lower than the dosage of thesubstance which is effective for production of another biologicaleffect. For example, it is shown below that administration ofmethoxsalen acted to increase plasma levels of nicotine and to reducedesire to smoke in dependent smokers at levels that were one-fourth thetherapeutic dose for treatment of psoriasis by methoxsalen.

The term “effective amount” as used herein means an amount effective andat doses and for periods of time necessary to achieve the desiredresults; this may mean limiting doses where the desired result isselective inhibition and selectivity is achieved through differentialinhibition of CYP2A. Preferably, the substances inhibit CYP2A6.

CYP2A6 Inhibitors

As hereinbefore mentioned, in one of its aspects, the present inventionrelates to a method of regulating nicotine metabolism to cotinine in anindividual comprising selectively inhibiting CYP2A6. Inhibition ofCYP2A6 may be achieved using one or more of the following (i) substanceswhich inhibit CYP2A6 activity; or (ii) substances which inhibittranscription and/or translation of the gene encoding CYP2A6.

Substances which inhibit CYP2A6 activity include substances whichspecifically bind to CYP2A6 and thereby inhibit its activity. Examplesof such substances include antibodies which are specific for CYP2A6including for example, the monoclonal antibody described by Pearce R, etal. (“Species differences and interindividual variation in livermicrosomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol,and testosterene oxidation,” Arch. Biochem. Biophys., 298(1): 211-225(1992)), and commercially available antibodies such as MAB2A6 andmonoclonal CYP2A6, sold by Gentest Corporation, Woburn, Mass., U.S.A.;XenoTech 2A6 sold by XenoTech LLC, Kansas City, Kans., U.S.A andpolyclonal CYP2A6 sold by Research Diagnostics, Inc, Flanders, N.J.,U.S.A.

Preferred inhibitors of CYP2A6 include methoxsalen, psoralen,tranylcypromine, pilocarpine, coumarin, chromone, esculetin, phenelzine,paroxetine, selegiline and pargyline.

Substances which inhibit CYP2A6 activity also include substances havinga lactone structure with a carbonyl oxygen. Non-limiting examples ofsuch substances include coumarin (The Merck Index, Eleventh EditionBudavari, S., ed. Merck & Co. Inc., 1989, No. 2563), furanocoumarin,methoxsalen (The Merck Index, No. 5911), imperatorin (The Merck Index,No. 4839), psoralen (The Merck Index, No. 7944), a-naphthoflavone,isopimpinellin, β-naphthoflavone, bergapten (The Merck Index, No. 1173),sphondin, coumatetralyl (racumin), and(+)-cis-3,5-dimethyl-2-(3-pyridyl)-thiazolidim-4-one (SM-12502) (Nunoya,et al., J Pharmacol. Exp. Ther., 277(2):768-74 (1996)). Other substanceswhich inhibit CYP2A6 and can be used in the methods and compositions ofthe invention include naringenin and related flavones,diethyldithiocarbamate, nicotine (useful primarily in the screeningmethods of the invention), N-nitrosodialkylamine (e.g.N-nitrosodiethylamine (The Merck Index, No. 6557),N-nitrosodimethylamine (The Merck Index, No. 6558)), nitropyrene,menadione (The Merck Index, No. 5714), imidazole antimycotics,miconazole (The Merck Index, No. 6101), clotrimazole (The Merck Index,No. 2412), pilocarpine (The Merck Index, No. 7395),hexamethylphosphoramide, 4-methylnitrosamine-3-pyridyl-1-butanol,aflatoxin B (The Merck Index, No. 168), tranylcypromine (the MerckIndex, No. 9491), including cis, trans, (+) and (−) isomers, trioxsalen,alaproclate, phenelzine, pargyline, paroxitine, selegiline, amphetamine,bupropion, buspirone, citalopram, desmethylcitalopram, doxeprine,fluoxetin, naltrexone, norfluoxetine, nortriptyline, sertraline,trazodone, viaqualine, zimelidine, chromone, bergapten and narigenin.All of the substances thati nhibit CYP2A6 activity include racemicmixtures of the compounds as well as the cis, trans, (+) and (−)isomers. See FIGS. 2A to 2D for the chemical structures of these andother non-limiting representative inhibitors. Selective andnon-selective monoamine oxidase inhibitors (e.g., alaproclate,phenelizine, deprenyl, pargyline, selegiline and the like) areparticularly preferred. Various isomers of the above compounds which canbe shown to inhibit CYP2A6 as described below are within thecontemplation of this invention.

Derivatives and analogs of these substances may also be used in themethods and compositions of the invention. Derivatives and analogsinclude compounds that are structurally similar to the compoundsdescribed herein and can bind to the CYP2A6 active site. For example,derivatives of tranylcypromine, coumarin and methoxsalen includepharmaceutically acceptable salts, esters and complexes oftranylcypromine, coumarin and methoxsalen including potassium and sodiumsalts, and amino acid, carbohydrate and fatty acid complexes. By way ofexample, suitable analogs of coumarin may be selected based upon theirfunctional similarity to coumarin, including the ability to inhibit themetabolism of nicotine to cotinine by CYP2A6. Examples of functionalanalogs of coumarin include 7-methoxycoumarin, 7-methylcoumarin, and7-ethoxycoumarin and all structures shown in FIGS. 2A, 2B, 2C. Analogsof coumarin may also be selected based upon their three dimensionalstructural similarity to coumarin—i.e., the lactone/carbonyl structure.

The present inventors have surprisingly found that natural products andextracts of natural products inhibit CYP2A6 activity in both human livermicrosomes and pure full-length human cDNA expressed cytochromes.Accordingly, CYP2A6 inhibitors of the present invention include naturalproducts or extracts of a natural product capable of inhibiting CYP2A6,such as Hypericum or a Hypericum extract or Cichorium intybus orBougainvllra spetabillis or an extract thereof.

The term “Hypericum” as used herein as synonymous with Hypericumperforatum, St. John's Wort, Goatweed and Klamath Wee. The phrase“Hypericum or an extract of Hypericum” as used herein includes the wholeplant Hypericum perforatum or a derivative, extract, isolate or purifiedcomponent thereof that can inhibit CYP2A activity. This includes naturalcomponents of the plant and synthetic analogues. A preferred extract ofHypericum is a methanol extract.

Derivatives of Hypericum which may be used in the methods andcompositions of the invention include hypericin, pseudohypericin,quercetin, hyperoside, quercitrin, isoquercitrin, rutin, campherol,luteolin, 13-II8-biopigenin, 1,3,6,7-tetrahydroxyxanthone,procyanidines, hyperforin, ethereal oil, phenol carbonic acids (e.g.chlorogenic acid), xanthone, phenylpropanes, flavonol derivatives,biflavones, proanthocyanidins, xanthones, phloroglucinols,naphthodianthrones and essential oil constitutes. Also included are thepharmaceutically acceptable salts, esters and complexes of thederivatives including potassium and sodium salts, and amino acid,carbohydrate and fatty acid complexes. Suitable derivatives of Hypericummay be selected based upon their ability to inhibit CYP2A with greaterthan 50% inhibition, and/or a Ki less than 300 μM.

The phrase “Cichorium intybus or Bougainvllra spectabillis or an extractthereof” as used herein includes the whole plants Cichorium intybus orBougainvllra specabillis or a derivative, extract, isolate or a purifiedcomponent thereof that can inhibit CYP2A activity. This includes naturalcomponents of the plants as well as synthetic analogues. A preferredextract from Cichorium intybus or Bougainvllra spectabillis isesculetin, esculin or esculin monohydrate.

Other extracts of natural products that may be useful in the presentinvention are shown in FIG. 20, and in U.S. provisional application Ser.No. 60/084,847, which is incorporated herein by reference.

The above lists of substances which inhibit CYP2A6 are provided by wayof example only and should not be seen as limiting the scope of thisinvention. Additional substances which inhibit CYP2A6 activity may beidentified using the screening methods described herein.

Substances which inhibit transcription and/or translation of the geneencoding CYP2A6 include a nucleic acid sequence encoding the CYP2A6 gene(GenBank Accession No. HSU22027) or parts thereof (e.g., the regionwhich is about 20 nucleotides on either side of nucleotide 790 (ATG),and the splice sites 1237, 2115, 2499, 3207, 4257, 4873, 5577 and 6308),inverted relative to their normal orientation for transcription—i.e.,antisense CYP2A6 nucleic acid molecules. Such antisense nucleic acidmolecules may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with CYP2A6 mRNA or the CYP2A6 gene. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

A nucleic acid molecule containing the antisense sequences may beintroduced into cells in a subject using conventional techniques, suchas transformation, transfection, infection, and physical techniques suchas electroporation or microinjection. Chemical methods such ascoprecipitation and incorporation of DNA into liposomes may also be usedto deliver antisense sequences. The molecules may also be delivered inthe form of an aerosol or by lavage. Suitable vectors or cloningvehicles for transferring the nucleic acid molecules are known in theart. Examples of suitable vectors include retroviral vectors, adenoviralvectors, and DNA virus vectors.

The ability of a substance to selectively inhibit CYP2A6 and thusregulate nicotine metabolism to cotinine may be confirmed using themethods described herein for screening for an inhibitor.

In one embodiment of the invention, the CYP2A6 inhibitor is at least onemember selected from the group comprising coumarin, methoxsalen,tranylcypromine, derivatives thereof and analogs thereof (see FIG. 2A).Initial in vitro screening and clinical studies have identified thatmethoxsalen is a potent inhibitor of CYP2A6.

CYP2A6 may also be selectively inhibited in the method of the inventionby interfering with the transcription of the gene encoding CYP2A6 usinggene transfer methods such as targeted gene mutagenesis using allelicreplacement, insertional inactivation, or deletion formation. Forexample, allelic gene exchange using non-replicating orconditionally-replicating plasmids has been used widely for themutagenesis of eukaryotes. Allelic exchange can be used to create adeletion of the CYP2A6 gene. Exemplary methods of making the alterationsset forth above are disclosed by Sambrook, et al. (1989).

CYP2B6 Inhibitors

CYP2B6 inhibitors may also be used in combination with inhibitors ofCYP2A6 to provide an enhanced inhibitory effect. Inhibitors of CYP2B6include one or more of the following (i) substances which inhibit CYP2B6activity; or (ii) substances which inhibit transcription and/ortranslation of the gene encoding CYP2B6. CYP2B6 inhibitors may also beused alone to inhibit nicotine metabolism in an individual.

Substances which inhibit CYP2B6 activity include substances whichspecifically bind to CYP2B6 and thereby inhibit its activity. Examplesof such substances include antibodies which are specific for CYP2B6including for example, commercially available antibodies such asanti-CYP2B6 sold by Gentest Corporation, Woburn, Mass., U.S.A.

Substances which inhibit CYP2B6 activity also include substancesselected from phenylethyl amines, diphenylbarbiturates, diethylsubstituted barbiturates and hydantoins. In particular, diphenhydramineand its derivatives, including orphenadrine (The Merck Index, No. 6831),and derivatives or analogs of orphenadrine, and other antihistamines,anticholinergic substances such as cholines and analogs and derivativesthereof may be used as CYP2B6 inhibitors in various embodiments of themethods and compositions of the invention. Antibodies, such aspolyclonal CYP2B1/2, polyclonal CYP2B1 and polyclonal CYP2B6 sold byGentest Corporation, Woburn, Mass., U.S.A., also bind specifically toCYP2B6 such that they also inhibit the activity of CYP2B6.

Derivatives of orphenadrine which may be used in the methods andcompositions of the invention include pharmaceutically acceptable salts,esters and complexes of orphenadrine including potassium and sodiumsalts, and amino acid, carbohydrate and fatty acid complexes. In oneembodiment, suitable analogs of orphenadrine may be selected based upontheir functional similarity to orphenadrine, including the ability toinhibit CYP2B6. Analogs of orphenadrine may also be selected based upontheir three dimensional structural similarity to orphenadrine.

Substances which inhibit transcription and/or translation of the geneencoding CYP2B6 include a nucleic acid sequence encoding the CYP2B6 gene(see FIG. 2B, GenBank Accession No. HSP452B6 for the mRNA sequence ofCYP2B6), or parts thereof (e.g., the region which is on either side ofnucleotide 9 (ATG), and the sites 111, 274, 424, 585, 762, 904, 1092,and 1234 nt), inverted relative to their normal orientation fortranscription—i.e., antisense CYP2B6 nucleic acid molecules. Suchantisense nucleic acid molecules may be produced and introduced intocells using conventional procedures as described herein.

CYP2B6 may also be selectively inhibited in a method of the invention byinterfering with the transcription of the gene encoding CYP2B6 usingconventional gene transfer methods as discussed herein.

In preferred embodiments of the invention the CYP2B6 inhibitor employedis orphenadrine and derivatives or analogs of orphenadrine.

An inhibitor of CYP2A6 or CYP2B6 may be targeted to the enzyme usingantibodies specific to an epitope of the enzyme. For example, bispecificantibodies may be used to target an inhibitor. The bispecific antibodiescontain a variable region of an antibody specific for at least oneepitope of CYP2A6 or CYP2B6, and a variable region of a second antibodywhich is capable of binding to an inhibitor. The bispecific antibodiesmay be prepared by forming hybrid hybridomas, using procedures known inthe art such as those disclosed in Staerz, et al. (“Hybrid hybridomaproducing a bispecific monoclonal antibody that can focus effectorT-cell activity,” Proc. Natl. Acad. Sci. USA, 83(5):1453-7 (1986)) andStaerz, et al. (Immunology Today, 7:241 (1986)). Bispecific antibodiesmay also be constructed by chemical means using conventional proceduressuch as those described by Staerz, et al. (“Hybrid antibodies can targetsites for a attack by T cells,” Nature, 314(6012):628-31 (1985)) andPerez, et al. (“Specific targeting of cytotoxic T cells by anti-T3linked to anti-target cell antibody,” Nature, 316(6026):354-6 (1985)),or by expression of recombinant immunoglobulin gene constructs.

Nicotine Replacement Therapy

An oral nicotine replacement therapy containing nicotine alone would beineffective due to the extensive metabolism of nicotine in the liverwhich significantly decreases the systemic availability of the nicotine.However, administering the nicotine with a CYP2A inhibitor wouldincrease the bioavailability and the effectiveness of the oral nicotinetherapy.

The present invention also includes a nicotine replacement therapycomprising contemporaneously administering to an individual in needthereof (a) oral nicotine and (b) one or more substances selected fromthe group consisting of (i) substances which inhibit CYP2A activity;(ii) substances which inhibit transcription, translation of the geneencoding CYP2A, or both; (iii) substances which delete all or a portionof the gene encoding CYP2A.

Preferably, the inhibitor is an inhibitor of CYP2A6 such as methoxsalenor tranylcypromine.

As used herein, “contemporaneous administration” of two substances to anindividual means providing each of the two substances so that they areboth biologically active in the individual at the same time. The exactdetails of the administration will depend on the pharmacokinetics of thetwo substances in the presence of each other, and can includeadministering the two substances within a few hours of each other, oreven administering one substance within 24 hours of administration ofthe other, if the pharmacokinetics are suitable. Design of suitabledosing regimens are routine for one skilled in the art, in view of thedetails provided herein on the biological activities of CYP2A6substrates and inhibitors. In particular embodiments, two substanceswill be administered substantially simultaneously, i.e., within minutesof each other, or in a single composition that contains both substances.On the other hand, a CYP2A6 inhibitor which acts by deleting or removingthe gene encoding CYP2A6 could be administered months or even yearsbefore administration of nicotine or a procarcinogen that wouldotherwise be converted to a carcinogen by CYP2A6, and the effects due tothe two administrations may still be contemporaneous.

Screening for Inhibitors

In addition to the CYP2A inhibitors listed above, substances which maybe used in the methods of this invention include other substances thatalter the kinetics for metabolism of nicotine to cotinine, alter smokingbehavior, alter the likelihood of addiction to smoking in a populationof non-smokers, alter the kinetics of formation for carcinogens whoseformation from procarcinogens is catalyzed by CYP2A. All of thesesubstances have in common an ability to reduce the activity of CYP2Aenzymes in an individual. The present disclosure therefore provides amethod of screening for a substance that inhibits a CYP2A enzyme in anindividual comprising assaying for a substance which selectively (i)inhibits CYP2A6 activity, (ii) inhibits transcription and/or translationof the gene encoding CYP2A6, or (iii) deletes or removes the geneencoding CYP2A6.

The inhibitory activity of a particular substance identified herein oran analog or derivative thereof may be confirmed by testing inexperimental model systems and in clinical studies, for example asoutlined below and exemplified in the Examples herein. Furthermore,specificity or selectivity of a substance listed above or a substancenewly identified by screening as described herein may be determined orconfirmed as described hereinbelow. While no particular test is mandatedby this invention, the usefulness of a particular substance (e.g., asubstance not specifically listed hereinabove or referred to in FIG.2A-2D) as a CYP2A6 inhibitor may be readily determining by testing thesubstance as follows.

In vitro Inhibition

An initial screen to select candidate inhibitors for use in the methodsaccording to this invention comprises:

(a) reacting, in the presence of a test substance, a substrate of CYP2A6with a source of CYP2A6 under conditions such that CYP2A6 is capable ofconverting the substrate into a reaction product;

(b) assaying for reaction product, unreacted substrate or unreactedCYP2A6;

(c) comparing the results of such assay to controls in the absence ofthe substance to determine if the test substance inhibits CYP2A6 andthereby is capable of inhibiting CYP2A enzymes.

Substrates of CYP2A6 which may be used in the in vitro test foridentification of substances for use in methods of the invention, aswell as in the in vivo tests below, include nicotine, coumarin, analogsthereof and derivatives thereof. The corresponding reaction products fornicotine and coumarin are cotinine and 7-hydroxycoumarin, respectively.

CYP2A6 used in the method of the invention may be obtained from natural,recombinant, or commercial sources. For example CYP2A6 may be obtainedby recombinant methods such as those described by Nesnow S, et al.(“N-nitrosodiethylamine and4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced morphologicaltransformation of C3H/10T1/2CL8 cells expressing human cytochrome P4502A6,” Mutation Research, 324:93-102 (1994)). Cells or liver microsomesexpressing CYP2A6 may also be used in the method.

Conditions which permit the formation of a reaction product may beselected having regard to factors such as the nature and amounts of thetest substance and the substrate. The results using the substrates inthe presence and absence of the test substance may be compared toresults using methoxsalen or tranylcypromine as controls which showpositive inhibition tests.

The reaction product, unreacted substrate, or unreacted CYP2A6; may beisolated by conventional isolation techniques, for example, salting out,chromatography, electrophoresis, gel filtration, fractionation,absorption, polyacrylamide gel electrophoresis, agglutination, orcombinations thereof.

To facilitate the assay of the reaction product, unreacted substrate, orunreacted CYP2A6; antibody against the reaction product or thesubstance, or a labeled CYP2A6 or substrate, or a labeled substance maybe utilized. Antibodies, CYP2A6, substrate, or the substance may belabeled with a detectable marker such as a radioactive label, antigensthat are recognized by a specific labeled antibody, fluorescentcompounds, enzymes, antibodies specific for a labeled antigen, andchemiluminescent compounds.

The substrate used in the method of the invention may be insolubilized.For example, it may be bound to a suitable carrier. Examples of suitablecarriers are agarose, cellulose, dextran, Sephadex, Sepharose,carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin,plastic film, plastic tube, glass beads, polyamine-methylvinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleicacid copolymer, nylon, silk, etc. The carrier may be in the shape of,for example, a tube, test plate, beads, disc, sphere etc. Theinsolubilized CYP2A6, substrate, or substance may be prepared byreacting the material with a suitable insoluble carrier using knownchemical or physical methods, for example, cyanogen bromide coupling.

In vivo Inhibition

Substances which pass the above-mentioned in vitro screening test arethen preferably subjected to an in vivo test to confirm theirsuitability for use in the methods of this invention. A suitable in vivotest method comprises the steps of:

(a) administering a subtherapeutic dose of nicotine (e.g., 1.0, 2.0 or4.0 mg expressed as the base) in an oral formulation to an individual,together with the test substance;

(b) collecting pre-nicotine and post-nicotine plasma samples from theindividual (e.g., 30, 60 and 90 minutes after (a));

(c) determining the plasma nicotine concentration using a conventionalanalytical technique (e.g., HPLC, gas chromatography and the like), and(d) comparing the plasma nicotine concentration to a control (i.e.,nicotine given without test substance) to assess whether the testsubstance results in a statistically significant increase in the plasmanicotine concentration at one or more time points, more preferably thelater time points.

Genetic Level Effectors

Analogous methods may be used for screening for a substance thatregulates nicotine metabolism to cotinine in an individual by inhibitingtranscription and/or translation of the gene encoding CYP2A6. Ascreening method for such substances comprises the steps of:

(a) culturing a host cell comprising a nucleic acid molecule containinga nucleic acid sequence encoding CYP2A6 and the necessary elements forthe transcription or translation of the nucleic acid sequence, andoptionally a reporter gene, in the presence of a test substance; and

(b) comparing the level of expression of CYP2A6, or the expression ofthe protein encoded by the reporter gene with a control cell transfectedwith a nucleic acid molecule in the absence of the test substance.

A host cell for use in the method of the invention may be prepared bytransfecting a suitable host with a nucleic acid molecule comprising anucleic acid sequence encoding CYP2A6. A nucleic acid sequence encodingCYP2A6 may be constructed having regard to the sequence of the CYP2A6gene (see the sequence under Genbank Accession number HUS22027,incorporated herein by reference) following procedures known in the art.Suitable transcription and translation elements may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate transcription and translationelements is dependent on the host cell chosen, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchelements include: a transcriptional promoter and enhancer or RNApolymerase binding sequence, a ribosomal binding sequence, including atranslation initiation signal. Additionally, depending on the host cellchosen and the vector employed, other genetic elements, such as anorigin of replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector. It will also be appreciated that thenecessary transcription and translation elements may be supplied by thenative CYP2A6 gene and/or its flanking sequences.

Examples of reporter genes are genes encoding a protein such asβ-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin, preferably IgG. Transcription of the reporter gene ismonitored by changes in the concentration of the reporter protein suchas β-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. This makes it possible to visualize and assay for expressionof CYP2A6 and in particular to determine the effect of a substance onexpression of CYP2A6.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,plant, or insect cells.

Protocols for the transfection of host cells are well known in the art(see, Sambrook, et al. (1989)). By way of example, Nanji M, et al.(“Expression in a baculovirus system of a cDNA encoding human CYP2A6,”Biochem. Soc. Trans., 22 (1994)) describe the expression of a cDNAencoding human CYP2A6 in a baculovirus system; Nesnow, S., et al. (1994)and Tiano H F, et al. (“Retroviral mediated expression of humancytochrome P450 2A6 in C3H/10T1/2 cells confers transformability by4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),” Carcinogensis,14:1421-7 (1993)) describe the expression of CYP2A6 from a retroviralvector in transformable C3H/10T1/2 mouse embryo fibroblasts; andSalonpaa P, et al. (“Retrovirus-mediated stable expression of humanCYP2A6 in mammalian cells,” Eur. J Pharmacol., 248:95-102 (1993))describe the preparation of amphotropic recombinant retrovirusescontaining CYP2A6 using LXSN vector and PA317 packaging cells.

Host cells which are commercially available may also be used in themethod of the invention. For example, the h2A3 (now known as h2A6) andh2B6 cell lines available from Gentest Corporation are suitable for thescreening methods of the invention.

Substances which pass the in vitro screening test for alteration ofexpression of CYP2A6 preferably are then subjected to an in vivo test toconfirm their suitability for use in the methods of this invention, byanalogy to the in vivo test for inhibitors of CYP2A enzyme activity.

The above mentioned methods may be used to identify negative regulatorsof nicotine metabolism to cotinine in brain and liver thereby affectingconditions requiring regulation of nicotine metabolism. Furtherconfirmation of the suitability of the substances, and/or demonstrationof the selectivity of the effects, may be achieved by population studiesof the effects of the substances on the kinetics for metabolism ofnicotine to cotinine, on smoking behavior, on the likelihood ofaddiction to smoking in a population of non-smokers, and/or on thekinetics of formation for carcinogens whose formation fromprocarcinogens is catalyzed by CYP2A. Such studies are a routine matterfor the skilled clinician in view of the guidance provided herein andthe exemplary studies described in the Examples below.

Compositions

Substances which inhibit CYP activity described in detail herein, orsubstances identified using the methods of the invention may beincorporated into pharmaceutical compositions. Therefore the inventionprovides a pharmaceutical composition for use in treating a conditionrequiring a reduction in the activity of a CYP2A enzyme comprising aneffective amount of one or more substances which selectively inhibitCYP2A6, and a pharmaceutically acceptable carrier, diluent, orexcipient. In one of its aspects, the invention provides apharmaceutical composition for use in smoking prevention, smokingtreatment, smoking regulation, regulating carcinogen formation, cancerprevention and/or cancer treatment. A method of treatment using such acomposition is also provided. Further, the treatment methods andcompositions of the invention may also be used together with otheractive compounds, including such other active compounds which aresusceptible to CYP2A6-mediated metabolism leading to an inhibition orreduction in effectiveness of the other active compound.

Conditions requiring regulation of nicotine metabolism to cotinineinclude nicotine use disorders—i.e., dependent and non-dependent tobaccouse, and nicotine-induced disorders—i.e., withdrawal. The conditions maydevelop with the use of all forms of tobacco (e.g., cigarettes, chewingtobacco, snuff, pipes, and cigars) and with prescription medications(e.g. nicotine gum, nicotine patch, spray, pulmonary inhalation or otherforms). In particular, the pharmaceutical compositions and treatmentmethods of the invention may be used to diminish a subjects desire tosmoke and thereby alter smoking behaviour. The pharmaceuticalcompositions and treatment methods of the invention may also be usedtogether with other centrally active pharmaceutical compositions thatmodify smoking behaviour (e.g. bupropion (a.k.a. Wellbutrin®) in itsvarious formulations), to decrease the dose of the centrally activecomposition or to increase its effectiveness in the treatment of tobaccodependence.

The compositions and treatment methods of the present invention byregulating nicotine metabolism in an individual are highly effective.The methods and compositions maintain the behavioural components ofsmoking and modify them by reducing nicotine metabolism to cotinine. Anindividual with reduced nicotine metabolism following administration ofa composition of the present invention, will alter smoking behaviour andsmoke exposure because of modification of nicotine requirements. Themethods and compositions of the invention show patterns of reduction,more sustained abstinence, and lower tobacco smoke exposure thanobtained with prior art methods in particular those using nicotinedeprivation.

The behavioural component of smoking is particularly important in somegroups of individuals, and thus the methods and compositions of theinvention in modifying and maintaining behavioural components may beparticularly useful in reducing smoking in those individuals. Forexample, it has been found that behavioural components are significantin tobacco use by women. The present invention permits the developmentof behavioural learning on an individual/or group basis.

The compositions and treatment methods of the invention are alsoparticularly suited to regulate nicotine metabolism in individuals orpopulations having high levels of CYP2A6. For example, Caucasians inNorth America have high levels of CYP2A6. An individual or populationhaving a high level of CYP2A6 can be identified using our methods formeasuring CYP2A6.

The compositions and methods of the invention also have the advantage ofindividualization and flexibility in treatment duration. Thecompositions and treatment methods are particularly suitable forseverely dependent individuals, previous treatment failures, individualsunable to accept the current approach of complete cessation,treatment/prevention of relapse, or concurrent treatment with othermethods such as the nicotine patch. It is expected that the compositionsand treatments of the invention will decrease the doses of nicotinepatch and all other forms of nicotine replacement therapies that areneeded and will prolong the duration of action of the therapy and/orenforce their effectiveness in the treatment of tobacco dependence.

The methods and compositions of the invention in treating individualswith nicotine use disorders and nicotine-induced disorders are alsouseful in the treatment and prophylaxis of diseases or conditions,including nicotine-related disorders such as opioid related disorders;proliferative diseases; cognitive, neurological or mental disorders; andother drug dependencies in the individuals. Examples of such underlyingdiseases or conditions include malignant disease, psychosis,schizophrenia, Parkinson's disease, anxiety, depression, alcoholism,opiate dependence, memory deficits, ulcerative colitis, cholinergicdeficits, and the like.

The methods and compositions of the invention may also be used in theprophylaxis and treatment of individuals having a condition whichrequires a reduction in CYP2A6 or CYP2B6. For example, CYP2A6 is knownto metabolize several procarcinogens such as NNK (Crespi C L, et al., “Atobacco smoke-derived nitrosamine,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiplehuman cytochrome P450s including the polymorphic human cytochromeP4502D6,” Carcinogenesis, 12(7):1197-201 (1991)), aflaxtoxin B1 (Yun CH, et al., “Purification and characterization of human liver microsomalcytochrome P-450 2A6,” Molec. Pharmacol., 40(5):679-85 (1991));hexamethylphosphoramide (Ding X, et al., “Mossbauer studies on themetal-thiolate cluster formation in Fe(II)-metallothionein,” Eur. JBiochem., 171 (3):711-4 (1988)), and nitrosodimethylamine (Davies R L,et al., “Development of a human cell line by selection anddrug-metabolizing gene transfection with increased capacity to activatepromutagens,” Carcinogenesis, 10:885-891 (1989); Fernandez-Salguero, etal. (1995)). Therefore, inhibitors of CYP2A6 may be useful in theprophylaxis (e.g., inhibition of CYP2A6 substrates thereby decreasinggenotoxicity, cytotoxicity and/or mutagenicity) and treatment ofmalignant diseases, and, without limitation, the above-mentionedconditions and diseases.

Formulation and Dosing

The pharmaceutical compositions of the invention contain substanceswhich inhibit CYP2A described in detail herein or substances identifiedusing the methods of the invention. The active substances can beadministered alone, but are generally administered with a pharmaceuticalcarrier etc. (see below), selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage administered will vary depending on the use and known factorssuch as the pharmacodynamic characteristics of the particular substance,and its mode and route of administration; age, health, and weight of theindividual recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired.

In some instances, instead of increasing the dosage of a compound, thekinetics of inhibition created by certain chemical compounds can bealtered or enhanced by adding to the treatment protocol a secondinhibitor to a substance (e.g., enzyme) that is capable of inhibitingthe metabolism of the CYP2A6 inhibitor. By adding such a secondinhibitor, the quantity of the CYP2A6 inhibitor will be maintained thusprolonging the beneficial effect of maintaining an elevated plasmaconcentration of nicotine. The use of such a second inhibitor is verybeneficial since it facilitates treatment of individuals by maintainingsubstantially constant nicotine levels and acting locally on thekinetics of the CYP2A6 inhibitor. By using this approach, large dosagesof centrally active compounds can be avoided.

Similarly, preexposure of an individual to an inhibitory substancesometimes can result in an inhibitory effect that will outlast thepresence of the drug in the plasma or that will have a persistent effectin the individual despite the inhibitor's half life in the plasma. Thisphenomenon caused by preincubation or preexposure of an inhibitorysubstance can help increase the dose interval at which a dosage of thesubstance must be administered, decrease the chronic dose or enhanceCYP2A6 inhibition. Furthermore, preexposure of an individual to oneinhibitory substance can subsequently decrease the needed dose of asecond inhibitor.

The appropriate dosage of a substance which selectively inhibits CYP2A6is dependent upon the amount of CYP2A6 that is present in anindividual's body. This amount is in turn dependent upon whether theindividual contains two mutant alleles, one mutant allele or no mutantalleles at the CYP2A6 gene locus. In Example 1, we confirmed that suchvariations can exist in the genetic material of a population. It is,therefore, an aspect of this invention to provide a method fordetermining the CYP2A6 activity in an individual containing two mutantalleles, one mutant allele or no mutant alleles at a gene locus for theCYP2A6 gene, the method comprising the steps of:

(a) assaying a bodily sample containing deoxyribonucleic acid (i.e. a“DNA-containing bodily sample”) from the individual to determine whetherthe individual contains two mutant alleles, one mutant allele or nomutant alleles at the CYP2A6 gene locus;

(b) determining the amount of CYP2A6 present in the individual; and

(c) correlating the results of assaying in step (a) and the amount ofCYP2A6 in step (b) to determine an appropriate dosage for thatindividual of a substance which (i) selectively inhibits CYP2A6activity, or (ii) selectively inhibits transcription and/or translationof the gene encoding CYP2A6.

The individual recipient may be any type of mammal, but is preferably ahuman. Generally, the recipient is an individual having a CYP2A6genotype associated with an active form of the enzyme. The CYP2A6genotype of an individual and the existence of an active CYP2A6 enzymein an individual may be determined using procedures described herein.For example, coumarin 7-hydroxylation has been used to measure CYP2A6activity (Cholerton, et al. (1992); and Rautio, et al., (1992)). Asdiscussed above, the methods and compositions of the invention may bepreferably used in individuals or populations having high levels ofCYP2A6, or in individuals where the behavioural components of smokingare significant.

For use in the treatment of conditions requiring regulation of nicotinemetabolism to cotinine, by way of general guidance, a daily oral dosageof an active ingredient such as coumarin or methoxsalen can be about0.01 to 80 mg/kg of body weight, preferably 0.01 to 20, more preferably0.05 to 3 mg/kg of body weight. Ordinarily a dose of 0.03 to 50 mg/kg ofcoumarin, methoxsalen or tranylcypromine per day in divided doses one tomultiple times a day, preferably up to four times per day, or insustained release form is effective to obtain the desired results. Inaccordance with a particular regimen, coumarin or methoxsalen ortranylcypromine is administered once to four times daily for as long asnecessary. While standard interval dose administration may be used thecompositions of the invention may be administered intermittently priorto high risk smoking times, e.g., early in the day and before the end ofa working day.

More than one substance described in detail herein or identified usingthe methods of the invention may be used to regulate metabolism ofnicotine to cotinine. In such cases the substances can be administeredby any conventional means available for the use in conjunction withpharmaceuticals, either as individual separate dosage units administeredsimultaneously or concurrently, or in a physical combination of eachcomponent therapeutic agent in a single or combined dosage unit. Theactive agents can be administered alone, but are generally administeredwith a pharmaceutical carrier selected on the basis of the chosen routeof administration and standard pharmaceutical practice as describedherein.

The substances for the present invention can be administered for oral,topical, rectal, parenteral, local, inhalant or intracerebral use. In anembodiment of the invention, the substances are administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using forms of transdermal skin patches known tothose of ordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will becontinuous rather than intermittent throughout the dosage regimen. Thesubstances can also be administered by way of controlled or slow releasecapsule system and other drug delivery technologies.

For example, for oral administration in the form of a tablet or capsule,the active substances can be combined with an oral, non-toxic,pharmaceutically acceptable, inert carrier such as lactose, starch,sucrose, glucose, methyl cellulose, magnesium stearate, dicalciumphosphate, calcium sulfate, mannitol, sorbitol and the like; for oraladministration in liquid form, the oral active substances can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Suitablebinders, lubricants, disintegrating agents, and colouring agents canalso be incorporated into the dosage form if desired or necessary.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Suitable lubricants used inthese dosage forms include sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride, and thelike. Examples of disintegrators include starch, methyl cellulose, agar,bentonite, xanthan gum, and the like.

Gelatin capsules may contain the active substance and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar carriers and diluents may be used tomake compressed tablets. Tablets and capsules can be manufactured assustained release products to provide for continuous release of activeingredients over a period of time. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration may contain colouring and flavouring agents toincrease patient acceptance.

Water, a suitable oil, saline, aqueous dextrose, and related sugarsolutions and glycols such as propylene glycol or polyethylene glycols,may be used as carriers for parenteral solutions. Such solutions alsopreferably contain a water soluble salt of the active ingredient,suitable stabilizing agents, and if necessary, buffer substances.Suitable stabilizing agents include antioxidizing agents such as sodiumbisulfate, sodium sulfite, or ascorbic acid, either alone or combined,citric acid and its salts and sodium EDTA. Parenteral solutions may alsocontain preservatives, such as benzalkonium chloride, methyl- orpropyl-paraben, and chlorobutanol.

The substances described in detail herein and identified using themethods of the invention can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

Substances described in detail herein and identified using the methodsof the invention may also be coupled with soluble polymers which aretargetable drug carriers. Examples of such polymers includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyl-eneoxide-polylysinesubstituted with palmitoyl residues. The substances may also be coupledto biodegradable polymers useful in achieving controlled release of adrug. Suitable polymers include polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels. The substances can also be affixed torigid polymers and other structures such as fullerenes or Buckeyballs.

Pharmaceutical compositions suitable for administration contain about 1milligram to 1500 milligrams of active substance per unit. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5-95% by weight based on the totalweight of the composition.

Suitable pharmaceutical carriers and methods of preparing pharmaceuticaldosage forms are described in Remington's Pharmaceutical Sciences, MackPublishing Company, a standard reference text in this field.

Co-administration with Oral Nicotine

In a particular embodiment, it has been found that specific inhibitorsof CYP2A6, preferably methoxsalen and/or tranylcypromine, areparticularly effective inhibitors of CYP2A6 and of the metabolism of anoral formulation of nicotine and as such, enhance the effect of oralnicotine replacement therapies. In other words, it has been found thatthese inhibitors are effective in inhibiting nicotine metabolism andthereby increasing plasma concentrations of nicotine, particularly whenthe nicotine is orally ingested thereby enhancing oral nicotinereplacement therapies.

Thus, this invention provides a composition for enhancing the effect oforal nicotine replacement therapy, comprising an inhibitor of CYP2A6 andnicotine formulated for oral ingestion. In this method, the substancesdescribed in detail herein and/or identified using the screening methoddescribed above, together with nicotine, form the active ingredient, andare typically administered in admixture with suitable pharmaceuticaldiluents, excipients, or carriers suitably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, consistent with conventionalpharmaceutical practices.

Those of skill in the art will recognize that oral formulation withinthe invention can be in the form of: (i) a single composition comprisingboth the CYP2A6 inhibitor and nicotine, or (ii) a kit comprisingindependently administered compositions comprising the CYP2A6 inhibitorand nicotine, respectively. For independently administered compositions,the administration is preferably substantially contemporaneous. When thepreferred CYP2A6 inhibitors methoxsalen and/or tranylcypromine areadministered with oral formulations of nicotine the plasmaconcentrations of nicotine have increased over the plasma concentrationswhen nicotine is orally digested without administering the CYP2A6inhibitor(s).

Combination of Inhibitors

The combination of an CYP2A6 inhibitor (e.g., coumarin, methoxsalen),and a CYP2B6 inhibitor (e.g., orphenadrine) enhances inhibition ofnicotine metabolism to cotinine. Thus, a preferred embodiment of theinvention provides a method for treating conditions requiring regulatingnicotine metabolism to cotinine comprising administering an effectiveamount of a CYP2A6 inhibitor and an effective amount of a CYP2B6inhibitor to selectively inhibit nicotine metabolism to cotinine. In apreferred embodiment of the invention, the CYP2A6 inhibitor ismethoxsalen or an analog or derivative thereof, and the CYP2B6 inhibitoris orphenadrine, or an analog or derivative thereof. The inhibitors maybe administered concurrently, separately or sequentially. Preferably,the administration of the inhibitors is substantially contempraneous.The doses of the CYP2A6 inhibitor and the CYP2B6 inhibitor are eachselected so that each inhibitor alone would not show a full effect. Theeffective doses are those which are approximately the minimum dosesadequate for enhanced inhibition of nicotine metabolism to cotinine. Inone mode, the combination of inhibitors may be administeredsubstantially contemporaneously with a source of nicotine, preferablynicotine formulated for oral administration. Pharmaceutical compositionscontaining combinations of CYP2A6 and CYP2B6 inhibitors may be prepared,and administered as described herein for the compositions containingCYP2A6 inhibitors. The pharmaceutical compositions preferably containmethoxsalen or an analog or derivative thereof, and orphenadrine, or ananalog or derivative thereof, in concentrations of 1 to 1500 mg, and 25to 400 mg, respectively.

Embodiments of the present invention will be illustrated with referenceto the following examples which should not be construed as limiting thescope of the invention.

EXAMPLES Example 1

Epidemiology Study

We examined the prevalence of CYP2A6 gene mutations in 126 tobaccodependent Caucasian smokers and 143 Caucasian individuals who had triedsmoking, but who had never became tobacco dependent smokers (e.g.,exposure controls). The objectives were two fold. The first was todetermine the incidence of individuals who were deficient in CYP2A6activity (e.g., homozygous for null CYP2A6 alleles). The second was todetermine if slower CYP2A6 mediated nicotine metabolism, due to havingnull CYP2A6 alleles, decreased the chances of becoming a tobaccodependent smoker.

In this Example a study was conducted to assess the CYP2A6 genotype in agroup of individuals and the effect of the CYP2A6 on the smokingbehaviour of the individuals.

Subjects were unrelated healthy individuals each with 4 Caucasiangrandparents and were divided into three groups. The first groupcomprised tobacco Dependent only (TD, DSM-IV (“DSM”=DiagnosticStatistician Manual of the American Psychiatric Association)) subjectsincluding 76 males aged 19 to 52 years old (mean (SD): 31.1 years old(8.5 years)), and 57 females aged 20 to 70 years old (mean (SD): 31.4years old (10.2)). The second group comprised Alcohol and TobaccoDependent (AT, DSM-IV) subjects including 60 males aged 17 to 61 yearsold (mean (SD): 37.2 years old (9.94 years)), and 10 females aged 19 to66 years old (mean (SD): 41.4 years old (11.89 years)). The third groupwas an exposure control group consisting of Never-Tobacco Dependent(NTD( subjects, who had previously tried smoking, but had never becomedependent. This group included 86 males 19 to 59 years old (mean (SD):29.2 years old (8.6 years)), and 77 females 19 to 58 years old (mean(SD): 27.4 years old (8.4 years)). All subjects completed a drugquestionnaire and tobacco module (Heatherton, et al., “The FagerstromTest for nicotine Dependence: a revision of the Fagerstrom ToleranceQuestionnaire,” Br. J. Addict., 86(9):1119-1127 (1991)). All subjectshad no other psychoactive drug dependencies, including alcohol (exceptof course for the AT group).

CYP2A6 genotyping of each subject was performed on genomic DNA isolatedfrom peripheral leukocytes as described by Fernandez-Salguero, et al.(1995). Briefly, the assay consisted of a CYP2A6 gene-specific nestedPCR amplification followed by a RFLP analysis.

Materials and Methods:

Primers Used for PCR Genotyping Assays:

TABLE 2 Assay Name Sequence (5′-3′) CYP2A6*2(v₁) F4CCTCCCTTGCTGGCTGTGTCCCAAGCTTAGGC (SEQ ID NO: 1) and R4CGCCCCTTCCTTTCCGCCATCCTGCCCCCAG (SEQ ID NO: 2) CYP2A6*3(v₂) E3FGCGTGGTATTCAGCAACGGG (SEQ ID NO: 3) E3R TCGTGGGTGTTTTCCTTC (SEQ ID NO:4)CYP2A6 Genotype

DNA is extracted from blood samples and quantified using routineextraction procedures. CYP2A6 genotype was determined using nested PCRand RFLP as described by Fernandez-Salguero, et al. (1995). The firstamplification, which is CYP2A6 gene-specific, was used to increase thespecificity for the CYP2A6 gene (versus other CYP2A genes). Exon 3 wasutilized in the second amplification because both the CYP2A6*2 andCYP2A6*3 mutant alleles contain nucleotide changes leading to amino acidchanges in this region of the CYP2A6 gene.

The first amplification was performed using the XL-PCR kit (Parkin-ElmerCo., Norwalk, Conn.). A 100 μl reaction mixture of 0.2 μM of primer F4and R4, 200 μM dNTPs, 0.8 mM magnesium acetate, and 2 U of rTth1 DNApolymerase and 400 to 600 ng of genomic DNA used. The amplification wasperformed in a MJ DNA Engine (MJ Research, Inc., Watertown, Mass.) at93° C. for 1 minute, 66° C. for 6 minutes and 30 seconds for 31 cycles.

The second amplification was performed in a reaction mixture containing0.5 μM of primers E3F and E3R, 200 μM dNTPs, 1.5 mM MgCl₂, 2.5 U of TaqDNA polymerase (Gibco BRL, Life Technologies, Burlington, Ontario), and2.5 μl of first amplification product, which was the template for thereaction. The reaction conditions were as follows: 94° C. for 3 minutes,followed by 31 cycles of 94° C. for 1 minute, 60° C. for 1 minute and72° C. for 1 minute.

The second amplification yielded a PCR product 201 bp in length whichwas digested with Xcm I (New England Biolabs) and Dde I (New EnglandBiolabs and Pharmacia Biotech) to detect the CYP2A6*2 and CYP2A6*3mutations, respectively (cutting indicates the presence of themutation). Concentrations of enzymes and PCR product, total volume anddigestion time were determined empirically to optimize cuttingefficiency with a minimal amount of time and enzyme. Xcm I digestionreactions were carried out at 37° C. for 2 hours in a 30 μl reactionmixture containing 1×NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mMMgCl₂, 1 mM DTT pH 7.9 @ 25° C.), dH₂O, and 2 U of Xcm I. Dde Idigestions were carried out at 37° C. for 2 hours in a 30 μl reactionmixture containing One-Phor-All (OPA) buffer (Pharmacia Biotech) and 2 Uof Dde I. Digestion products were analysed on ethidium-stained 3%agarose gels.

Blood samples were obtained under consent from all subjects. Positivecontrols were donated by Drs. P. Fernandez-Salguero and H. Raunio.Negative controls used water in place of genomic DNA. Every genotypingreaction carried four randomly selected samples from a previous reactionto check for reproducibility.

Chi-Square tests were performed comparing distribution of CYP2A6genotypes and alleles between groups (SAS). F-tests (check for unequalvariance [SAS]), followed by a two-sample t-test (SAS) were used incomparing smoking patterns within smokers. Significance was at 5%.

It was postulated that individuals with impaired nicotine metabolism(i.e., carriers of at least one CYP2A6 defective or mutant allele) wouldexperience greater aversive effects due to higher nicotine levels andnot become smokers or would smoke at a decreased level when compared toindividuals with active nicotine metabolism (i.e., individuals havingCYP2A6*1/CYP2A6*1 genotype). Specifically, it was hypothesized thatthere would be an under-representation of individuals carrying defectiveCYP2A6 alleles in a tobacco dependent population.

With reference to the results of the study, among the total dependentsmokers (TD+AT), the frequency of individuals carrying 1 or 2 of theCYP2A6 defective alleles was lower than in the exposure control group(NTD): 13.3% vs. 20.2%, p=0.076, c-square; Odds Ratio of 1.66, C.I.0.95-2.89—see FIG. 5. Further, both the CYP2A6*2 and CYP2A6*3 allelefrequencies were lower in the dependent smokers (TD and AT) than in theexposure control group (NTD): CYP2A6*2: 3.0% versus 3.1% and CYP2A6*3:4.4% versus 7.7%—see FIG. 5.

We further postulated that, within the group of those who smoke, thosewith deficient nicotine metabolism (i.e., carriers of at least oneCYP2A6 defective or mutant allele) would smoke fewer cigarettes.

With further reference to the results of the study, within the TD group,those subjects which were homozygous for CYP2A6 active alleles smokedsignificantly more cigarettes per day and per week when compared withsmokers who were heterozygous carrying a single CYP2A6 defective allele(i.e., one or both of CYP2A6*2 and CYP2A6*3): 23 versus 19 cigarettesper day, t test P=0.02, 125 versus 161 cigarettes per week, t testP=0.009—see FIG. 6.

As discussed above, nicotine is important in establishing andmaintaining tobacco dependence; variability in nicotine pharmacokineticscould have a profound influence on whether individuals become smokers.The data produced in this study demonstrates an under-representation ofindividuals carrying 1 or 2 of the CYP2A6 defective alleles in a tobaccodependent population (TD+AT) when compared to a never tobacco dependent(NTD) control population. While not wishing to be bound by anyparticular theory or mode of action, this may be caused when individualswho carry CYP2A6 defective alleles, upon smoking, experience highernicotine levels and greater aversive effects to the nicotine. As aresult, the individual may discontinue smoking and be less likely tobecome tobacco dependent. Therefore, the data produced in this studyindicates that individuals who carry 1 or 2 of the CYP2A6 defective ormutant alleles, and who try smoking, are at lesser risk for becomingtobacco dependent than individuals who have two active CYP2A6 alleles.

Further, as discussed above, it is well known that dependent smokersadjust their smoking behaviour in order to maintain blood and brainnicotine concentrations, and thus, variable nicotine metabolism couldplay a role in altering smoking patterns. The observation that dependentsmokers who carry a single defective CYP2A6 allele smoke significantlyfewer cigarettes when compared to homozygous wild-type smokers indicatesthat nicotine metabolism, as mediated by CYP2A6, is a significantdeterminant in the amount that dependent smokers smoke. In other words,heterozygosity in a single gene, namely the CYP2A6 gene, is affectingthis complex drug taking behaviour.

The clinical implications of CYP2A6 genotype on tobacco dependence andsmoking behaviour disclosed herein are widespread. Individuals who carryCYP2A6 defective alleles may have a decreased risk for cancerdevelopment because they have a decreased risk of becomingtobacco-dependent smokers. If they do become dependent smokers, the dataproduced in this study demonstrates that they would smoke less thanthose homozygous for active CYP2A6 alleles. There is clear evidence thatthe amount of tobacco smoked is related to increased risk for lungcancer (Law, et al. 1997))—see FIG. 6.

In addition, tobacco smoke contains a number of tobacco specificprocarcinogen nitrosamines, such as N-nitrosodialkylamines—e.g.,N-nitrosodiethylamine (the Merck Index, No. 6557),N-nitrosodimethylamine (The Merck Index, No. 6558) and4-methylnitrosamino)-1-)3-pyridyl)-1-butanone (Crespi, et al. 1990;Yamazaki, et al. 1992). As these procarcinogens can be activated byCYP2A6, individuals who carry CYP2A6 defective alleles will beadvantageously inefficient at bioactivating tobacco smoke procarcinogensto carcinogens.

Thus, in summary the data produced in the study of this Exampledemonstrates that a single genetically polymorphic gene, the CYP2A6gene, is related, and in some cases predictive, of whether an individualbecomes a smoker. In addition, if an individual becomes dependent ontobacco alone, the CYP2A6 gene variants alter the number of cigarettesthat he/she smokes. Accordingly, the CYP2A6 genotype directly influencesthe risk for tobacco dependence, alters the amount of tobacco consumed,and plays a role in tobacco-related cancer susceptibility.

One envisaged application of the present invention is the geneticidentification of an individual's risk for smoking and related cancers.Identification of high and low risk individuals will allow targetedprevention, treatment and education. Specifically this will involve theidentification of an individual with a high and low risk for: (i)becoming a smoker (if the individual is a non-smoker), (ii) highertobacco-consumption (if the individual is a smoker), and (iii)CYP2A6-related cancers.

Example 2

Coumarin Phenotyping Test and CYP2A6 Genotyping Test

A. Coumarin Test

Coumarin is a selective and specific substrate for human CYP2A6 and canbe used to: (1) identify individuals who are potential therapeuticexclusions for use of CYP2A6 inhibitors; (2) for dosage refinement basedon the initial level of activity of CYP2A6; and (3) for risk factorassessment in identifying individuals who will not benefit from thetreatment or who may be at risk to toxicity from agents which areinhibitors and substrates themselves of CYP2A6. The Coumarin Test existsin two forms:

(1) Coumarin Test When Only Urine is Available

Coumarin 5 mg formulated in a capsule or other dose form is administeredorally to fasted individuals after voiding of residual bladder urine.Urine is collected for the first 2 hours and for the subsequent 6 hours.The amount of urinary excretion of the coumarin metabolite 7hydroxy-coumarin (free and conjugated) is determined by determining theconcentration of these metabolites on the urine using an HPLC assay asdescribed in an earlier example. The relative activity of CYP2A6 isreflected in the total amounts of 7 hydroxy-coumarin excreted in thesampling periods separately and combined and the activity can beexpressed as the ratio of the percent coumarin excretion (amountexcreted in the first 2 hours/amount excreted in 8 hours)×100. Thispercent excretion ranges from values less then 20% in individualswithout CYP2A6 activity to >80% in individuals with high activity. Thistest can be equally effectively and reliably be applied to smokers andnon-smokers and may be used at any time of day with out apparent effectof the smoking condition or time of day on the results. The testdemonstrates high within subject reproducibility with a linear rof >0.9. See FIG. 3 for results of a study in which smokers andnonsmokers were given coumarin in the morning and afternoon on each of 2separate days. High within subject reproducibility and reliability isdemonstrated.

(2) Coumarin Test When Plasma Samples Can Be Taken

In some clinical situations blood samples can be easily taken or arenecessary as part of other clinical tests. In this situation, aplasma-based test of CYP2A6 activity has been developed and applied toindividuals of known genotype. Individuals ingest coumarin 5.0 mg orallyand 45 minutes later a blood sample is drawn in a heparinized (or otheranticoagulant containing tube). The sample is spun and the plasmaseparated. The plasma is analysed by HPLC to quantitate 7hydroxycoumarin (total after deconjugation with beta glucuronidaseincubation). High analytical sensitivity is required in order to use 5.0mg of coumarin. When such sensitivity is not available, the dose ofcoumarin may be increased up to 50 mg.

HPLC Analysis of 7-hydroxycoumarin in Urine and Plasma:

(1) Sample Preparation:

Urine or plasma samples (0.5 ml) are hydrolyzed with 0.2 ml ofβ-glucuronidase acetate buffer solution (15 mg/ml acetate buffer, 0.2 M,pH 5.0) at 37° C. for 30 min. Extraction is followed with 2 ml ether byvortex for 5 min and centrifuged at 3000 rpm for 10 min. Ether extract(1.2 ml) is transferred to another clean tube and dried down undernitrogen gas. The residue is reconstituted in the HPLC mobile phase (seebelow), and injected onto HPLC.

(2) HPLC Analysis:

The HPLC system consists of Hewlett Packard 1050 HPLC system (pump,autosampler and UV detector) and HP3396II integrator. Thechromatographic separation was performed with an HP Spherisorb-ODS2column (125×4 mm I.D., 5 μm). Samples were eluted with a mobile phase ofacetonitrile:water:acetic acid of 150:850:2 (v/v/v) at a flow rate of1.0 ml/min, and monitored by a UV detector at a wavelength of 324 nm for7-hydroxycoumarin and 280 nm for coumarin. Samples are quantitativelydetermined by an external standard method.

The CYP2A6 activity is expressed as the concentration of 7hydroxy-coumarin in the plasma at various points in time (e.g. 20, 30,45 and 75 minutes) or as the ratio of coumarin/7 hydroxy-coumarin in theplasma at that time.

The preferred mode of use is a simple plasma sample at 20 or 30 minutesafter the oral administration of coumarin in which both coumarin and7-hydroxycoumarin are quantified and in which the coumarin to7-hydroxycoumarin ration is used as the index of CYP2A6 activity.

Results:

Blank urine or plasma samples showed no interfering peak for7-hydroxycoumarin or coumarin. Sensitivity of this method is 1 ng/mlurine or plasma. Intraday and inter-day variations are less than 10%.This analysis is linear from 1 ng to 4000 ng/ml.

FIG. 4 is a graph showing a time course of total 7-hydroxycoumarinconcentration detected in the plasma of subjects given coumarin. FIG. 4illustrates various time courses based on corresponding genotypes forCYP2A6.

B. CYP2A6 Genotyping Test

As for the CYP2A6 genotyping test, mutant alleles which decrease CYP2A6activity in an individual can be screened in a DNA sample using thematerials and screening method described in Example 1.

Example 3

In Vivo Phenotype Assay

The plasma kinetics of nicotine and coumarin were compared after oraladministration in 10 smokers and 9 non-smokers (12 males, 7 females) ofknown CYP2A6 genotype. The dose of nicotine was 4.0 mg (expressed asbase) and the dose of coumarin was 50 mg. The plasma concentration ofnicotine, cotinine, coumarin and 7-OH-coumarin were measured asdescribed above.

Optimal separation of *1/*1 (wild type homozygotes, n=13) andheterozygotes (*1/*2; *1/*3, n=4) and homozygotes (*2/*2, n=2) was foundat 45 min with coumarin by measuring its metabolite 7-OH-coumarin(7-OH-coumarin [μM] *1/*1=5.6±2.9; *1/*2 or *1/*3−3.8±1.1, p=0.04).Optimal separation was found at 90 min with nicotine (nicotine [nM]*1/*1=24±15; *1/*2 or *1/*3=29±12; *2/*2=52±3; *2/*2 vs. *1/*2 or *1/*3,p=0.01; *2/*2 vs. *1/*1, p=0.0001). The use of thecoumarin/7-OH-coumarin or nicotine/cotinine ratio did not improveseparation.

Cotinine (nicotine metabolite) was significantly more slowly produced in*1/*2 or *1/*3 initially, but late in sampling cotinine was actuallyhigher in *1/*2 or *1/*3, suggesting a role for CYP2A6 in cotininemetabolism. The slope of the curve for appearance vs. time wassignificantly less for 7-OH-coumarin and greater for nicotine in *1/*2or *1/*3 compared to *1/*1, as were the areas under the curves,indicating differences in bioavailability, rates of absorption andmetabolism. The area of the curves for 7-OH-coumarin and for nicotinewere inversely correlated (p=0.08 (Spearman rank), n=18). One *1/*1individual with 4-fold greater 7-OH-coumarin production and one *2/*2individual with high coumarin and low nicotine metabolism wereidentified, suggesting that CYP2A6 gene variants not detected withcurrent PCR procedures exist.

These data indicate CYP2A6 genotype is an important determinant of timecourse for nicotine disposition in vivo. These data also indicate thatan in vivo assay of CYP2A6 reflects the phenotype of the individual, andthat the phenotypic determination provides information on the genotypeof that individual.

Example 4

Tissue Localization of CYP2A6 Expression

It was determined if the CYP2A6 enzyme was expressed in tissues in whichtobacco-related cancers occur (e.g., lung and bladder). In order todetermine whether CYP2A6 was expressed in tissues which were subject totobacco-related cancers, the tissue distribution of the CYP2A6 mRNA wasexamined in various human tissues using Northern blot analysis. BrieflymRNA from numerous tissues was loaded onto gels and separated byelectrophoresis. The mRNA was then transferred to membranes and probedwith radioactive CYP2A6 cDNA probe. Evidence was found for theexpression of CYP2A6 in uterus, ovaries, colon, small intestine, testis,bladder, heart, stomach, prostate, skeletal muscle, pancreas and lung.These data suggest that in addition to the possibility thatCYP2A6-activated carcinogens from the liver might cause cancer invarious tissues there could also be in situ activation of theprocarcinogens in a number of human tissues.

Example 5

Epidemologic Study for Cancer Risk

In addition to the role that null CYP2A6 alleles have in reducing therate of nicotine-dependence and the amount smoked if one becomesdependent, procarcinogens found in tobacco-smoke can be activated byCYP2A6. In order to determine whether there was a significantcontribution to cancer rates due to activation of procarcinogens byCYP2A6, independent of its role in smoking, a study was made ofindividuals with a tobacco-related cancer who were non-smokers. Theseindividuals were passively exposed to tobacco smoke procarcinogens,therefore providing a group in which the role of the CYP2A6 null allelesin the activation of procarcinogens could be tested independently of therole of this enzyme on smoking behaviour. It was found that 14.3% ofnon-smokers who had bladder cancer (a tobacco-related cancer) carried anull allele for CYP2A6. In contrast, in the control population who werenon-smokers and did not have bladder cancer, the frequency of nullallele carriers was 24.1%.

This demonstrates that individuals who carry null alleles for CYP2A6 areless likely to get a tobacco-related cancer due to their decreasedactivation of procarcinogens to cancer-causing carcinogens.

Example 6

Effect of Methoxsalen, a CYP2A6 Inhibitor, on Activation of NNK, aProcarcinogen, to its Metabolites NNAL and NNAL Glucuronide

The effect of methoxsalen, CYP2A6 inhibitor, on nicotine metabolism andNNAL production was studied in eleven (n=6 females, 5 males) tobaccodependent smokers. Subjects were recruited only if they smoked at least15 cigarette per day, and were required to smoke the same number ofcigarettes each day, on all four study days. During assessment, bloodwas taken to be analyzed for plasma nicotine and cotinine levels, aswell as for CYP2A6 genotyping. Breath carbon monoxide was also measured.

The four test days were broken down into one placebo day (no drug given)(day 1) and three methoxsalen (10 mg t.i.d. p.o.) treatment days 2, 3,4), such that medication was taken at 8:00 a.m., 3:00 p.m. and 10:00p.m. each day. During each study day a smoking log was completed. Thislog asked subjects to document the number of cigarette smoked from 8:00a.m. to 3:00 p.m. to 10:00 p.m., and 10:00 p.m. to 8:00 a.m. Study days1 and 2 could be separated, but days 2, 3 and 4 were required to beconsecutive.

On both study days 1 (placebo) and 4 (treatment), a 24 h urinecollection was initiated on waking. At 2:00 p.m. blood was drawn fornicotine and cotinine analysis and breath carbon monoxide was measured.On day 4, a second blood sample was taken for methoxsalen analysis.Urine samples can be analyzed for 24 h NNAL, NNAL glucuronide,creatinine and cotinine.

Methoxsalen increased plasma nicotine by 17% (23.0 to 27 ng/ml);decreased breath CO by 11% (22.5 to 20.5 ppm) and increased the ratio ofplasma nicotine to breath CO (an index of tobacco smoke exposure) by 30%(1.1 to 1.43, p=0.033). The larger decrease in the index of smokeexposure indicates: 1) the smokers decreased the intensity of theirsmoking due to inhibition of CYP2A6 and slowed nicotine eliminationdespite being told to not change their smoking; 2) methoxsalen in thesedoses is an effective inhibitor of CYP2A6; and 3) methoxsalen and otherCYP2A6 inhibitors will decrease production of NNAL or related substancesand the activation of other carcinogens in vivo.

Example 7

Influence of the CYP2A6 Null Alleles on Tobacco-smoke Exposure Leadingto Lung Cancer

The effect of the CYP2A6 null alleles on activation of procarcinogenswas examined in an epidemiological study of lung cancer. The allelefrequencies in DNA samples from 227 individuals with lung cancer wasdetermined. Following diagnosis of lung cancer and resection of thetumor and surrounding lung tissue, the DNA was extracted and genotypedfor CYP2A6. The population was principally smokers or ex-smokers. Amongthose from whom detailed smoking histories were available, we were ableto assess the number of pack-years of smoking (20 cigarettes/day for oneyear=one pack-year) as a measure of procarcinogen exposure, prior todetection of the lung cancer. Those individuals who had wt/wt CYP2A6activity required an average of only 45 pack-years prior to lung cancerdetection. In contrast, those individuals with a CYP2A6 null allele, whoactivate less of the procarcinogens, required considerably moreprocarcinogen exposure prior to detection of lung cancer (e.g., 54pack-years). Thus those individuals with decreased CYP2A6 activityactivate less of the tobacco-smoke procarcinogens and require greaterexposure before lung cancer is detected.

It was also observed that the number of individuals with the CYP2A6V1allele in the lung cancer population was decreased relative to anon-lung cancer control population, consistent with the relativeprotection against cancer offered by decreased procarcinogen activation.This was observed in both non-smokers and smokers with lung cancerrelative to their respective controls.

In addition, all of the individuals who had ras oncogene mutations inthe tumor tissues were full activity CYP2A6 wildtype indicating adecreased risk for oncogene mutations in those individuals withdecreased CYP2A6 activity (e.g., carriers of the CYP2A6 null alleles).Ras oncogene mutations are predictive of a poorer treatment outcome andfaster growing tumors relative to those tumors without ras oncogenemutations. This suggests that individuals with CYP2A6 null alleles, wereless likely to have mutated ras oncogenes, and were more likely to havea better treatment prognosis.

These data indicate the utility in assessing CYP2A6 alleles forestimation of cancer risk (null alleles being associated with decreasedrisk for lung cancer) and that inhibition of CYP2A6 will decreaseprocarcinogen activation decreasing the risk for cancer.

Example 8

Effect of CYP2A6 Inhibition on the Bioavailability of Oral Nicotine

An in vivo study was undertaken to determine the effect of CYP2A6inhibition on the bioavailability of oral nicotine. In particular, thestudy compared the kinetic effects of nicotine cotreatment withmethoxsalen 30 mg, tranylcypromine 10 mg and of placebo (i.e., nicotineonly) p.o. on the bioavailability of nicotine 4 mg p.o (expressed asbase; nicotine bitartrate salt was actually administered), and the acutesafety and acceptability of the three cotreatments. Additionally,preliminary information was obtained about effects on nicotine cravingfollowing these cotreatments.

The subjects for the study were smokers following a specified abstinenceregimen as set out below. There were 12 subjects.

The smokers underwent placebo p.o. and two separate cotreatments 30(methoxsalen 30 mg and tranylcypromine 10 mg) accompanying nicotine 4 mgp.o. During a 4-hour test session, blood and urine samples werecollected for kinetic measures, and physiologic and subjective measureswere collected. The treatment order was randomized and counterbalancedand the drug presentations were single-blind, namely, the subjects wereunaware of what drug they were taking.

All of the subjects had the following characteristics:

-   -   (a) age at least 21;    -   (b) current consumption of at least 25 cigarettes per day;    -   (c) DSM IV current tobacco dependence;    -   (d) nicotine dependence as indicated by a score of at least 3 on        the Fagerström Test of Nicotine Dependence (FTND);    -   (e) no regular use of tobacco in any form other than cigarettes;    -   (f) ability to abstain from cigarette smoking and from caffeine        for up to 12 hours;    -   (g) agreement and ability to maintain the use of any therapeutic        drugs on a consistent schedule across the study days.

All of the subjects did not have any of the following characteristics:

-   -   (a) known sensitivity to methoxsalen or chemically similar        compounds;    -   (b) known excessive photosensitivity;    -   (c) current use of any antidepressants, sympathomimetics, CNS        depressants, hypotensive agents, or antiparkinsonian drugs        (because of possible adverse tranylcypromine interactions);    -   (d) body weight <51 kg (30 mg methoxsalen is not recommended for        use below this body weight);    -   (e) pregnancy or lactation;    -   (f) risk of pregnancy (females who are sexually active with male        partners and not using highly effective contraceptive        precautions, defined as surgical sterilization of either        partner, oral contraceptives, barrier and spermicide, or condom        and spermicide);    -   (g) liver damage, blood dyscrisias (counterindications for        tranylcypromine);    -   (h) symptoms suggestive of cardiac disease or hypertension;    -   (i) any other medical or psychiatric condition that requires        further investigation or treatment or that is a contraindication        for any of the proposed study drugs;    -   (j) current desire or attempts to quit smoking within the        expected duration of the study series; and    -   (k) any other condition likely to interfere with compliance with        the study schedule or successful collection of study measures.

There were two separate study schedules for subjects tested in themorning and in the afternoon. On both schedules, each subject abstainedfrom tobacco, food, beverages (i.e., other than water), and anyinconsistently used drugs from midnight before each study day butcontinued to take any regularly scheduled drugs allowed by the protocol(e.g., oral contraceptives, daily vitamins).

Subjects on the morning schedule continued such abstinence until arrivalat the test site at approximately 8 am. Subjects on the afternoonschedule ate a normal-sized breakfast, including at most one cup of acaffeinated beverage and one cigarette before 9 am. From 9 am then toapproximately 1 pm subjects in the afternoon session resumed theabstinence regimen until arrival at the test site.

Before baseline measures were taken, a breath CO sample (Ecolyzer) wastaken to assess compliance with the smoking abstinence (<10 ppmexpected). The subsequent daily schedule is set out in Table 3. Allmeasurements were with respect to a time zero at 9:00 am or 2:00 pm, atwhich time a nicotine capsule and a cotreatment are taken p.o. Each SMScycle consisted of heart rate, blood pressure, and subjective measures.A standard breakfast or lunch (but without caffeine) was served afterthe blood sample at +1:00 h.

TABLE 3 Time Elapsed Event −00:45  Prepare the subject −00:30  Bloodsample (8 mL) taken - #1 −00:15  SMS cycle #1 00:00 All capsules p.o.00:30 Blood sample (8 mL) taken - #2 00:50 SMS cycle #2 01:00 Bloodsample (8 mL) taken - #3 01:30 Blood sample (8 mL) taken - #4 01:50 SMScycle #3 02:00 Blood sample (8 mL) taken - #5  2:50 SMS cycle #4 03:00Blood sample (8 mL) taken - #6

Subjects were medically assessed for discharge no earlier than +2:00 andwere discharged after all measures were completed at +3:00 h.

Subjects were not allowed to smoke until after their discharge. Theschedule was in certain circumstances delayed by up to 20 minutes,consistent across the four days, in order to allow three subjects to betested on the same day.

Each subject was tested on two non-consecutive days in a week for threeseparate weeks and maintained a morning or afternoon scheduleconsistently.

Sterile nicotine bitartrates was obtained by the Pharma Centre fromSigma Chemical. The reported purity was >99.5% which was confirmed byHPLC. The nicotine bitartrate powder was measured on a precision balanceaccurate to within 1 μg, measured into portions containing 4 mg of thenicotine base, and then encapsulated. Capsules were filled to atolerance of +2% of their nominal mass of nicotine powder.

Methoxsalen is marketed in Canada in two forms, one of relatively lowbioavailability (trade name Oxsoralen®) and two of approximately twiceas high a bioavailability (Oxsoralen-Ultra® and Ultra MOP®). The packageinsert for Ultra MOP®) (Canderm Pharmacal Ltd.) recommends a daily doseof 30 mg to 50 mg for any patient weighing 51 kg or more. Subjects wererestricted to a minimum body weight of 51 kg, in order to allow the useof a fixed 30 mg dose for all subjects; the dose was not adjusted forlarger subjects.

Capsules of methoxsalen 10 mg and tablets of tranylcypromine 10 mg(Parnate®) were used in their marketed forms. Because it was arandomized but singleblind study, subjects were able to recognize thatthe capsule forms and number varied from day to day, but they did notknow which form represented which drug.

Lactose tablets were used for the placebo.

For the first four days, each day's drug supply consisted of one orthree capsules, as set out in Table 4, in addition to nicotine 4 mg. Theinvestigators were unaware of the distribution to preserve randomallocation.

The nicotine and other capsules were taken simultaneously.

A separate, printed, reference copy of each subject's treatmentrandomization code was provided to the investigators after the drugswere dispensed.

TABLE 4 Active Drug, Capsule Form Administered Dosage placebo 1 ×coumarin-size placebo methoxsalen, 30 mg 3 × methoxsalen, 10 mgtranylcypromine, 10 mg 1 × tranylcypromine, 10 mg

Using an indwelling venous catheter, 8 mL blood samples were collectedat the elapsed times set out in Table 3. These samples were analyzed fornicotine, cotinine and inhibitor concentration.

Two separate urine samples were collected, a baseline just prior to thecapsules and a three-hour pooled sample. Each urine sample was analyzedfor nicotine and cotinine content.

Plasma and urinary nicotine and cotinine (and also the conjugates inurine) were determined using an HPLC method with a UV detector.Specifically, 1 mL of sample, 50 μL (2 μg/mL) of the internal standard(N-ethylnornicotine) and 1 mL of trichloroacetic acid (10%) werepipetted into each tube (12 mL). The tube was capped, vortex-mixed for afew seconds, and then centrifuged at 30,000 g for 5 minutes. The clearsupernatant was decanted in a second tube. To this protein-free plasmaextract was added 0.5 mL of a 5 M potassium hydroxide solution and 6 mLof methylene chloride. The second tube was then capped, agitated for 30minutes in a horizontal shaker and then centrifuged to separate thephases. The aqueous phase (the top layer) was aspirated, and 3.0 mL of0.5 N hydrochloric acid solution was added to the organic phase andvortex-mixed for 30 seconds. The phases were separated bycentrifugation, and the aqueous phase was transferred to a clean tubewith 0.5 mL of 5 M potassium hydroxide solution, followed by addition of5 mL of methylene chloride and vortex mixing for 30 seconds. The phaseswere separated by centrifugation, the aqueous (top) layer was aspirated,and 200 ml methanolic hydrochloric acid (10 mmol HCl in methanol) wasadded to the remaining solution and mixed gently. The organic solventwas then evaporated under nitrogen in a water bath at 40° C. The sidesof the tube were washed with 200 μL of methanolic hydrochloric acid andthe solution was evaporated. The residue was reconstituted in 100 μL of30% methanol and 90 μL thereof was injected in the HPLC column.

The chromatographic separation was performed with a Supelco™ 5-8347LC-8-DB (150×4.6 mm, 5 μm). The sample was eluted with a mobile phase of0.34 M citric acid buffer:acetonitrile, 800:45 (v/v) containing 0.34 MKH₂PO₄, 1-heptane sulphonate (671 mg) and triethylamine (5 mL) with aflow rate of 1.3 mL/min, and monitored by a UV detector at I=260 nm.

The sensitivity of the nicotine assay is <1 ng/mL and that of thecotinine is <5 ng/mL. Conjugates in urine were determined afterhydrolysis with β-glucuronidase, when appropriate.

Plasma nicotine concentration was determined using the above-mentionedassay, and the results are illustrated in FIG. 7 as the mean for allsubjects.

Cardiovascular measures, transduced by a Hewlett/Packard 78352C AdultPatient Monitor and recorded directly into a computer included heartrate and blood pressure (while seated).

Using a visual analog scale, each subject was asked to evaluate, on ascale of 0 to 100, his/her current desire to smoke a various points intime, both pre- and post drug administration (Appendix A).

For the purposes of establishing clinical kinetic differences anddescribing kinetic parameters, the primary dependent variable was the3-hour trapezoidal-rule nicotine AUC.

FIG. 7 illustrates the mean plasma nicotine concentrations measured justprior to oral drug administration and for three hours thereafter (duringwhich no smoking was allowed). As illustrated, the combinedmethoxsalen/nicotine and tranylcypromine/nicotine treatments both inducean increase in mean plasma nicotine concentration that is at least fourtimes as large as that induced by the placebo/nicotine combination.

FIG. 8 illustrates the self-rated “Current desire to smoke” evaluation,using a visual analog scale scored from 0 to 100. As illustrated, boththe methoxsalen/nicotine and tranylcypromine/nicotine combinationreduced the desire to smoke significantly more than does theplacebo/nicotine combination.

Example 9

Effects of Metabolically Enhanced Oral Nicotine Replacement Therapy onShort-term Smoking Behaviour

A study was undertaken to determine the effects of metabolicallyenhanced oral nicotine replacement therapy on short-term smokingbehaviour.

Because nicotine is the addictive agent in tobacco dependence, andsmokers regulate their brain nicotine within a fairly narrow individualconcentration band, any effective non-smoking method of nicotinedelivery should result in a decrease in smoking by allowing smokers tomaintain plasma nicotine without resorting to smoking and, in somecases, reduce the secondary reinforcement of smoking behaviour as acomponent of eventual smoking cessation. This effectiveness can only beenhanced if the mechanism for ensuring effective delivery also delaysthe clearance of plasma nicotine after the absorption stage. A model fortesting the acute behavioural effects of this treatment strategy isprovided in a previous study of the effectiveness of nicotine gum(Nemeth-Coslett R, et al., “Nicotine gum: dose-related effects oncigarette smoking and subjective ratings,” Psychopharmacology,92(4):424-30 (1987)), where smoking in a 90-minute test period wassignificantly affected by the nicotine content of the gum.

In this Example, all four combinations of placebo/methoxsalen andplacebo/nicotine were tested (i.e., (i) placebo methoxsalen andnicotine; (ii) methoxsalen and nicotine, (iii) methoxsalen and placebonicotine; and (iv) placebo methoxsalen and placebo nicotine).

This Example demonstrates: the kinetic effectiveness ofmethoxsalen-enhanced oral nicotine replacement therapy in brieflyabstinent smokers; and the behavioural effectiveness ofmethoxsalen-enhanced oral nicotine replacement therapy in brieflyabstinent smokers.

There were 11 subjects. The subject inclusion and exclusion criteriaused in Example 8 were used in this Example.

The subjects each underwent four separate sessions of 90 minutes smokingabstinence followed by 90 minutes ad lib smoking, where the followingfour treatments were each presented double-blind once during the firstfour study days, in randomized, counterbalanced order, during theabstinence period: (i) placebo methoxsalen with placebo nicotine, (ii)placebo methoxsalen with 4 mg nicotine, (iii) methoxsalen 30 mg withplacebo nicotine, and (iv) methoxsalen 30 mg with 4 mg nicotine.Methoxsalen 10 mg capsules were used, as in Example 1, and capsules ofroyal jelly were used as the placebo. Capsules were dispensed in anopaque vial, and neither subjects nor investigators viewed the capsulesprior to the subjects placing them directly into their mouths.

Nicotine 4 mg capsules were prepared as in Example 8, and correspondingroyal jelly capsule placebos containing only lactose were also prepared.Subjects took the three methoxsalen/placebo capsules and onenicotine/placebo capsule either all at once or consecutively.

The treatment order was counterbalanced across the two sexes and the twotimes of day to the extent possible. The order of the treatments wasdetermined by a computerized randomization program. The treatment wasdouble-blind.

As in Example 8, there was a morning and afternoon schedule.

Study sessions were scheduled twice each day, with three subjectsrunning simultaneously, beginning at 8:30/8:40/8:50 am and at1:00/1:10/1:20 pm. Sessions lasted for about 4 hours. Prior to eachsession, subjects were allowed to smoke, eat, and drink caffeine as theydesired up to 90 minutes before each session, at which time they stoppedeating and drinking (other than water). Each session began 60 minutesbefore a drug/placebo/nicotine were taken. The precise schedule is setout in Table 5.

TABLE 5 Time Elapsed Event −00:40  Pre-abstinence cigarette −00:30 Abstinence begins −00:10  1. Blood sample (8 mL) taken - #1 2. COmeasured 3. Questions 1-9 in Appendix C 00:00 1. CO measured 2. Allcapsules p.o. 00:50 1. Blood sample (8 mL) taken - #2 2. CO measured 3.Questions 1-12 in Appendix C 00:59 1. CO measured 2. Video camera on01:00 1. Abstinence ends 2. Free smoking begins 02:30 1. Video cameraoff 2. Free smoking ends 3. Blood sample (8 mL) taken - #3 4. COmeasured 5. Questions 1-17 in Appendix C 6. Abstinence resumes 02:40 COmeasured 02:50 CO measured 03:00 CO measured

The first post-cigarette abstinence period lasted 90 minutes, of whichthe last 60 minutes were post administration of placebo/drug/nicotine.The second abstinence period was included to facilitate repeatedmeasures of the post-smoking breath carbon monoxide (CO). On the threeoccasions when blood samples were collected, the subject also answered abrief questionnaire about possible study drug symptoms and effects andabout desire to smoke. Additionally, on the third occasion, there werealso questions about perception of the cigarettes smoked during the freesmoking period. This questionnaire (see Appendix A) is based on the oneused in the nicotine gum study (Nemeth-Coslett, et al. (1987)).

During the free smoking period, subjects were allowed to smoke as theywished, providing only that they stay within the area of the roomvisible to the video camera, and to eat light snacks and drinknon-caffeinated beverages.

The primary dependent variable measured was the change in breath COduring the free-smoking period, measured as the mean of the threesamples 10, 20, and 30 minutes post-smoking minus the mean of the twosamples 10 and 0 minutes pre-smoking. Other dependent variables measuredwere the change in plasma nicotine between 0 and 150 minutes post-drug,the responses to the symptom and rating scales, the consumption oftobacco in the free smoking period (measured as the weight of buttsremaining subtracted from the weight of the same number of unsmokedcigarettes), and the puff timing and count from analysis of thevideotapes.

Dependent variables were evaluated in an analysis of variance, with thetreatment drug combinations as the primary independent variable ofinterest, with sex, morning/afternoon schedule, and treatment order asadditional explanatory variables removed from the error term.

The results of the study of Example 10 will now be discussed withreference to FIGS. 9-14.

FIG. 9 illustrates the mean breath carbon monoxide concentrationmeasured just prior to oral drug administration, 60 minutes later (nosmoking allowed) and after the 90 minute free smoking period. Asillustrated, the combined methoxsalen/nicotine treatment results in asignificant and large reduction in the increased in breath carbonmonoxide during the smoking phase. The carbon monoxide levels increasedin the combined methoxsalen/nicotine treatment group only 30% of thatseen in the other conditions, reflecting a large reduction in smokingand smoke exposure. This reduction may be attributable due to anycombination of fewer puffs, shallower puffs and/or puffs held for ashorter duration before exhalation.

FIG. 10 illustrates the ratio of the increased plasma nicotineconcentration to increased breath carbon monoxide concentration over the90 minute free smoking period. In other words, this Figure illustratesthe measure of potential reduction in smoke exposure that might occurwhile dependent smokers replenish their systemic plasma nicotinecontent. As illustrated, the methoxsalen/nicotine treatment stands apartfrom all three of the other treatments and from their mean, more thandoubling the gain in nicotine per unit of increase in breath carbonmonoxide concentration.

FIG. 11 illustrates the commonly used measure of smoking: the meannumber of cigarettes smoked during the 90 minute period. As illustrated,the combined methoxsalen/nicotine treatment is associated with the leastsmoking, however number of cigarettes consumed is an insensitive measureof smoke exposure and smoking behaviour compared to breath carbonmonoxide concentration.

FIG. 12 illustrates the mean cumulative number of puffs taken by the endof each 10 minute period during the 90 minute free smoking period, andthe data is summarized by the area under this curve. This area wouldincrease if either the total number of puffs increased, or if the samenumber were consumed earlier thereby shifting the curve to the left. Asillustrated, the methoxsalen/nicotine treatment is associated with thefewest cumulative number of cigarette puffs.

FIG. 13 illustrates that the number of grams of tobacco burned issignificantly less in the methoxsalen/nicotine treatment group. Again,this measure is less sensitive than direct measures of smoking behaviourand smoke exposure (e.g., breath carbon monoxide concentration andnicotine/breath carbon monoxide concentration).

FIG. 14 illustrates that the inhibition of CYP2A6 metabolism of nicotineachieves the reduction in breath carbon monoxide by changingnicotine-regulated smoking behaviour. The latency between cigarettes issignificantly prolonged by the combined methoxsalen/nicotine treatment.

In summary, the results illustrated in FIGS. 9-14 show a modification insmoking behaviour by subjects who were treated with a combination ofmethoxsalen and nicotine. Specifically, key objective indicators such asplasma nicotine concentration and breath carbon monoxide weresignificantly reduced compared to the other treatment regimens.

CYP2A6 metabolizes approximately 75% of nicotine in vivo.Tobacco-dependent smokers regulate their smoking to maintain nicotinelevels; CYP2A6 inhibition should decrease nicotine metabolism, decreasesmoking and smoke exposure (e.g. breath CO). Overnightnicotine-abstinent dependent smokers (6 males, 5 females) smoked onecigarette followed by one of four oral drug combinations in crossovercounter-balanced order: methoxsalen 30 mg (CYP2A6 inhibitor K; =0.2 μM)or placebo with either nicotine 4.0 mg or placebo. Sixty minutes later,subjects started 90 mins of ad libitum smoking. Subjects receivingmethoxsalen with oral nicotine smoked less than in the placebo/placebo(e.g. breath CO 50% less increase; latency to the second cigarette 83%increase, number of cigarettes smoked 24% decrease; tobacco burned(grams) 24% decrease, total number of puffs taken decrease 25% [allp<0.05]). In addition, on several measures (e.g. latency to secondcigarette) the rank order of response wasmethoxsalen/nicotine>methoxsalen/placebo>placebo/nicotine>placebo/placebosuggesting a methoxsalen effect on systemic clearance of nicotine.CYP2A6 inhibition alone or combined with oral nicotine decreases smokingand could have a role in tobacco smoking cessation, exposure reductionor relapse prevention strategies.

Example 10

Inhibitors Of Nicotine's Metabolism: Potential New Treatments ForTobacco Dependence

Nicotine is inactivated by CYP2A6 to cotinine. As nicotinebioavailability is low (20-35%), an oral nicotine replacement isfeasible. In vitro inhibition of nicotine metabolism was studied inexpressed CYP2A6 using tranylcypromine, methoxsalen, and as inhibitors(K_(i)=0.05-6 μM). In dependent smokers methoxsalen, 30-50 mg orally 30min prior to nicotine 31 μg/kg subcutaneously (3 doses, hourly),increased the 8-hour mean plasma nicotine by 49% (p<0.01) while coumarin(225 mg hourly×6) increased it by 15% (p<0.05) compared to placebo.Using methodology described above, studies conducted in regular smokers(n=7-12) given nicotine (4.0 mg p.o.) alone and concurrently withplacebo, methoxsalen (3 mg, 10 mg or 30 mg) or tranylcypromine (2.5 mgor 10 mg) significantly increased the plasma nicotine compared toplacebo (see FIG. 15). Specifically, methoxsalen 10 and 30 mg (M10 andM30, FIG. 15) and tranylcypromine 2.5 and 10 mg (T2.5 and T10, FIG. 15)produced approximate 100% increases in plasma nicotine (p<0.01). Inaddition, these increases in oral nicotine bioavailability wereassociated with a significant (p<0.05) decrease in desire to smoke forM10 and M30 and T2.5 (p=0.17) (see FIG. 15, solid circles).

From these studies it is evident that methoxsalen and tranylcypromine indoses ¼ and ⅛, respectively, of their current therapeutic doses used fortreating psoriasis and depression, respectively, can be used to inhibitnicotine first-pass metabolism and systemic metabolism. Other CYP2A6inhibitors and their isomeric forms can be expected to do the same.

Example 11

Extraction of Hypericum

The following procedures were used to prepare an extract of Hypericumperforatum:

An extraction mixture of 10 Hypericum capsules (0.3% hypericin) and 50ml of 80% methanol (water 20%, v/v) was placed in a beaker (125 ml). Inone protocol the extraction was carried out using cold methanol. In asecond protocol, the extraction mixture was placed in a water-bath andheated until boiling (about 80° C.). The mixture was kept boiling for 30min, keeping the volume to 50 ml by adding methanol, then cooled down atroom temperature, and centrifuged at 3,000 g for 5 min. The resultingextract was then blown to dryness and resuspended in trisbuffer pH 7.4.

Example 12

Effect of Hypericum Extracts on CYP2A6 Activity

Nicotine metabolism was monitored in vitro. Inhibitor assessment wascarried out by comparing the assay including 50 μl tris buffer with theextracts prepared in Example 11 (50 μl, diluted in tris buffer). Humanliver microsomes or CYP2A6 microsomes were used. The concentration ofinhibitor in Hypericum was calculated on the basis of an apparentmolecular weight of the inhibitor of 504 and a concentration of 0.3%active material in the plant material when diluting to yieldconcentrations of 20, 10, 5, 1, 0.1 and 0.01 and 0.01 μM.

Incubation Mixture:

-   -   Substrate 80 μM (50 μl tris buffer)    -   NADPH1 mM (50 μl tris buffer)    -   Microsomes 80 μg (30 μl tris buffer)    -   Cytosol (rat) 20 μl (tris buffer)    -   Tris buffer 50 μl (pH7.4)    -   Final volume 200 μl        Incubation Procedure:

The incubation was carried out at 37° C. for 30 min. An internalstandard (50 μl, caffeine, 0.05 mg/ml) was added to the incubationmixture, followed by DCM (1 ml). The mixture was shaken for 10 min andcentrifuged for 5 min at 3,000 g. The top layer was aspirated. HC1 (0.01N, 100 μl) was added, followed by shaking for 1 min, then centrifuge for5 min at 3,000 g. 30 μl of the HCl layer was injected for HPLC.

HPLC Conditions

-   Column: Supelco; 5-8347; LC-8-DB; 150×4.6 mm; 5 μm-   I: 260 nm-   Flowrate: 1.3 ml/min-   Mobile phase: 1000/120 (citric buffer/acetonitrile, v/v)-   Citric buffer: Citric Acid 7.14 g (0.34 M)    -   KH₂PO₄ 4.627 g (0.34 M)    -   1-Heptane sulphonate 670 mg    -   Triethylamine 5 ml    -   Final volume 1000 ml    -   Adjust the pH to 4.40 with 5 N KOH        Retention Times (min):    -   2.2 (nicotine iminium ion)    -   2.7 (nicotine)    -   3.4 (continine)    -   3.9 (caffeine) (internal standard)

FIG. 16 demonstrates the inhibition of nicotine metabolism with (1) coldmethanol extract of Hypericum; (2) a hot methanol extract of Hpericum;(3) purified Hypericin; and (4) a negative control. The results indicatethat both methanol extracts of Hypericum can inhibit CYP2A6 activity.

Example 13

Effect of St. John' Wort on Nicotine Metabolism

Twelve subjects received placebo or 3 St. John's Wort capsules 300 mgplus oral nicotine 4.0 mg as base orally. Blood samples were taken atdesignated times over 3 hours.

The results shown in FIGS. 18 and 19 demonstrate that St. John's Wortincreases the bioavailability of nicotine in vivo. The mean plasmanicotine concentrations were 64% higher after St. John's Wort.

Example 14

Effect of Esculetin on CYP2A6 Activity

Esculetin, a compound found in Cichorium intybus and Bougainvllraspectabillis, was tested for its ability to inhibit nicotine metabolismby CYP2A6 using the methods set forth in Example 12.

The results, shown in FIG. 19, demonstrate that esculetin is a potentinhibitor of CYP2A6.

Having illustrated and described the principles of the invention in apreferred embodiment, it should be appreciated to those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principles. We claim all modifications coming withinthe scope of the following claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Average Number of Cigarettes Smoked Per Day Genotype FrequencyTD AT NTD TD AT Males Females Total Males Females Total Age 28.3 31.237.8 31.1 31.4 31.2 37.2 41.4 37.8 wt/wt 130 114 62 26.1 19.5 23.2 28.927.8 28.7 wt/mut 31 17 7 19.3 18.7 19.1 29.7 50.0 32.6 mut/mut 2 2 122.5 — 22.5 25 — 25 n 163 133 70 76 57 133 60 10 70 wt/wt:CYP2A6*1/CYP2A6*1 wt/mut: CYP2A6*1/CYP2A6*2 + CYP2A6*1/CYP2A6*3 mut/mut:CYP2A6*2/CYP2A6*2

1. A method of stopping smoking of tobacco or reducing the amount oftobacco smoked comprising contemporaneously administering to anindividual in need thereof an effective amount of nicotine and aneffective amount of selegiline.
 2. The method according to claim 1,wherein said nicotine is formulated for oral administration.
 3. Themethod according to claim 1, wherein said selegiline and said nicotineare formulated in a single composition.
 4. The method according to claim1, further comprising administering an inhibitor of CYP2B6 to saidindividual contemporaneously with said selegiline and said nicotine. 5.The method according to claim 1, wherein said selegiline or nicotine areformulated for oral, topical, rectal, parenteral, local, inhalant orintracerebral administration.
 6. The method according to claim 5,wherein said topical administration is via a transdermal route.
 7. Themethod according to claim 1, wherein said nicotine is formulated fororal administration and said selegiline is formulated for topicaladministration.
 8. The method according to claim 7, wherein saidnicotine is formulated for topical administration and said selegiline isformulated for oral administration.
 9. The method according to claim 1,wherein said selegiline or nicotine are administered by way of acontrolled or slow release system.